Team:Freiburg bioware/Notebook/September

From 2009.igem.org

(Difference between revisions)
Line 79: Line 79:
<div class="art-Post-inner">
<div class="art-Post-inner">
<div class="art-PostMetadataHeader">
<div class="art-PostMetadataHeader">
-
<h2 class="art-PostHeaderIcon-wrapper">September<span
+
<h2 class="art-PostHeaderIcon-wrapper">October<span
  class="art-PostHeader"></span> </h2>
  class="art-PostHeader"></span> </h2>
</div>
</div>
Line 86: Line 86:
<div style="text-align: left;">
<div style="text-align: left;">
-
<h3>01.09.09, Manu, Hannes, Christoph, Laura</h3>
+
<h3>01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel</h3>
-
<br>*evaluation of sequencing
+
<br>* Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
-
- pMA_Shortlinker, plasmid prep from 29.08.09: sequence ok<br>
+
<br>* Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
-
- pEx_HisDigSplitFoka, plasmid prep from 29.08.09: sequence ok<br>
+
<br>* plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
-
- pEx_HisDigSplitFoki, plasmid prep from 29.08.09: sequence ok<br>
+
<br>* plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1
-
- pEx_StrepDigSplitFoka, plasmid prep from 29.08.09: sequence ok<br>
+
<br>* glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80°C
-
- pEx_StrepDigSplitFoki, plasmid prep from 29.08.09: sequence ok<br>
+
<br>
-
- pMA_ShortlinkerFoka, plasmid prep from 29.08.09: sequence not ok, the linker short middle and long were ordered without NgoMIV restriction sites -> has to be ordered again<br>
+
<br>* protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
-
- pMA_ShortlinkerFoki, plasmid prep from 31.08.09: see above<br>
+
<br>* SDS gel of protein purification HisFluASplitFoki
-
- pEx_HisFluASplitFoka, plasmid prep from 29.08.09: not Foka but Foki<br>
+
-
- pEx_HisFluASplitFoka, plasmid prep from 28.08.09: not Foka but Foki<br>
+
-
- pEx_HisFluASplitFoki, plasmid prep from 28.08.09: sequence ok<br>
+
-
- pEx_Strep-Dig: sequence ok<br>
+
-
- pEx_His-Dig: sequence ok<br>
+
-
- pMAFoka prep II from 05.08.09: sequence ok<br>
+
-
- pMAFoka prep II from 05.08.09: sequence ok<br>
+
-
- pMAFoki prep II from 05.08.09: sequence ok<br>
+
-
<br>*hybridization of M13 DNA ss and the fokcontrols in equal relations
+
-
<br>*made SDS gels --> 4°C
+
-
<br>*SDS gel of protein expression (pEx-His-FluA-SplitLi-Foki) from 28.08.09
+
-
[[Image:Freiburg09_010909_pg_pExHisFluASplitLiFokIvom2808_waehrend_expression012.jpg|none|thumb|SDS-Gel; pExHisFluASplitFoki; Lanes: NEB prestained protein marker, t0 (before induction), t1 (1 hour after induction), t2 (2 hours after induction), t3 (3 hours after induction), t4 (4 hours after induction)|400x400px]]
+
-
[[Image:Freiburg09_010909_pg_NiNTA_pExHisFluASplitLiFokIvom2808011.jpg|none|thumb|SDS-Gel; pExHisFluASplitFoki ; Lanes: NEB prestained protein marker, Elution fraction 1 (after NiNTA-column), Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 5, Elution fraction 6, Elution fraction 7, Elution fraction 8, Elution faction 9 |400x400px]]
+
-
Lane 4 showed some strange bands, so we repeated the gel<br>
+
-
<br>*made 1l buffer for AGO cleavage assay: 100mM NaCl, 10mM HEPES, 5mM MnCl2, pH 7.5
+
-
<br>*came up with idea for new BioBrick standart that would allow making fusiun proteins via "mega primer" pcr technic with only a Threonine as a scar and thereby skipping the cloning procedure:
+
-
[[Image:Freiburg 09 Superstandard.jpg|none|thumb||400x400px]]
+
-
The new restriction enzyme would be AcuI, that for example has no cutting site in M13mp18 and just one in pET28 and two times in pMAL-p5X core vector <br>
+
-
AcuI cuts 14/16 bases away from its recognition sequence end would thereby cut off the whole BB prefix and most of the BB suffix (with just AC left)
+
-
<br>*purification of pExHisFluASplitFoki via Ni-NTA column
+
-
<br>*inoculation of pExHisFluASplitFoki36GSlinker
+
-
<br>*starter cultures of
+
-
- pExHisDigSplitFoka<br>
+
-
- pExStrepDigSplitFoka<br>
+
-
in 50 ml LB and Ampicillin from plates of 31.08.09
+
-
<h3>02.09.09, Manu, Hannes, Christoph, Gerrit, Laura, Isabel</h3>
+
[[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel; HisFluASplitFoki; Lanes: NEB prestained protein marker, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, flow through fraction 2, washing fraction 2|400x400px]]
-
<br>*Inoculation of expression culture
+
-
- pExHisDigSplitFoka<br>
+
-
- pExStrepDigSplitFoka<br>
+
-
in 2 times 1 liter each at 28°C , 118rpm<br>
+
-
-> induction of expression at OD 0,4, harvesting 4 hours later<br>
+
-
-> centrifugation at 4000 rpm, 4°C, 20 min
+
-
-> put pellets in -80°C freezer
+
-
<br>*cleaning of Ni column with 15 ml water and 10 ml Ethanol (20%)
+
-
<br>*plasmid preparation of pExHisFluASplitFoki36GSlinker, glycerol stocks and sequencing
+
-
<br>*repetition of SDS gel of eluate fractions (pExHisFluASplitFoki)
+
-
see picture...
+
-
-> fractions 2 and 3 showed the probable Foki fusion protein and were united and dialysed
+
-
[[Image:Freiburg09_020909_pg_pExHisFluASplitLiFokIvom2808_repeat013.jpg|none|thumb|SDS-Gel; pExHisFluASplitFoki ; Lanes: NEB prestained protein marker, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 5, flow through fraction 1, flow through fraction 3, washing fraction 1 (250mM imidazole), washing fraction 3|400x400px]]
+
-
<br>*order of new short, middle and long linkers with XbaI, NgoMIV and AgeI restriction sites
+
-
<br>*made binding, washing and elution buffer (1 liter each) again for His/Ni purification
+
-
<br>*prepared 2 liters of dialysis buffer (30mM NaCl, 20mM Tris/HCl, pH 7,4)
+
-
<br>*centrifugation of the expression culture (20 min, 4000 rpm, 4°C), stored pellets at -20°C
+
-
<br>*started dialysis over night with 200 ml dialysis buffer and 3 ml protein solution (elutions 2 and 3 were combined), stiring at 4°C<br><br>
+
-
<b>Meeting</b><br>
+
<br>* pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
-
<br>*Kristian introduced GBM, please add your data to the list if you want to join the group founded for iGEM
+
<br>*phage display:
-
<br>*Fok
+
- desalt ligation products (for electroporation)<br>
-
- the tagged Oligo's arrived during the absence of Kristian and stayed one week in his mail box at room temperature. Christoph wanted to find out, how this affects the DNA<br>
+
pool samples (~80µl 445+87/89 and ~52µl 445+87/88)<br>
-
- we should repeat the Fok cutting assay with M13 dsDNA and change the origami-programm to stop at 37°C (instead of 20°C)<br>
+
add 1 volume of isopropanol, mix<br>
-
- new Oligo's for the cutting assay must be ordered with a length of 60 bp<br><br>
+
-80°C for 10 minutes<br>
-
- dialyse the his-tag purified protein solution to get the right ion concentrations for a ion exchange chromatography<br>
+
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
-
- with the his-tag purified and dialysed protein solution we must do some tests: freeze it, thaw it and measure the stray light(photometer)<br>
+
add 1 volume of 75% ethanol (without mixing)<br>
-
- prepare aliquots of the protein solution<br>
+
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant<br>
-
- do a purification via ion exchange chromatography (MonoQ, positive charged)<br>
+
let dry on heat block (50°C), lid open<br>
-
- try a first cutting assay with our construct<br>
+
add 25µl water<br>
-
- confirm the Oligo-Protein-interaction with a fluorescence or absorption measurement (we have to do some research on this)<br>
+
thermo shaker for 1h, 45°C, 1000rpm
-
<br>*Fok-Phage Display
+
-
- we want to assemble the different parts with a PCR with overlapping primers (including the GS-linker). We should use a mix of Taq and a proof reading polymerasy and have a maximum cycle number of 20 to keep the mutations as low as possible<br>
+
-
- order parts at Mr.Gene with iGEM restriction sites<br>
+
-
- we must plan the PCR-primers!!!<br>
+
-
<br>*AGO
+
-
- first try to cut single strand DNA<br>
+
-
<br>*book rooms in a hotel (Anika wanted get some info about the hotels)
+
-
<h3>03.09.09, Manuel, Hannes, Christoph, Gerrit, Timo</h3>
+
- PCR (4 samples each template):<br>
-
<br>*Hybridization of M13 ssDNA with FokKontrolle 2(1:100) and 3(1:100 dil)
+
buffer (with MgCl2): 5µl<br>
-
<br>*Digestion of M13 ssDNA FokKontrolle(FokK)1-8; 1&2;2&3;1&3 with FokI <br>
+
primer #7: 1,5µl<br>
-
M13 dsDNA with FokI. made a heat inactivation of 5b (95°C 10min) and 6b without FokI
+
primer #1: 1,17µl<br>
-
<br>*Cutting assay checked by gel electrophoresis<br>
+
dNTPs: 2µl<br>
-
[[Image:Freiburg09_09.09.03_gelcuttingassay2.JPG|none|thumb|Agarose gele of M13DNA digest with fokI: Marker(fermantas gene ruler mix), M13ssDNA+fokI, M13ssDNA fokk1+fokI, M13ssDNA fokk2+fokI, M13ssDNA fokk3+fokI, M13ssDNA fokk2/3+fokI, M13ssDNA fokk2/3+fokI(heatshocked), M13ssDNA fokk1/2+fokI, M13ssDNA fokk1/2, M13ssDNA fokk2/3+fokI(37°C), M13dsDNA fokk2/3+fokI|400x400px]]
+
Taq: 1µl<br>
-
<br>*Digestion of pEX_His_FluA_Split_Foki_36GSli(prep 02.09.09 rest4 tube3) with AgeI and PstI<br>
+
MnCl2: 0,5µl<br>
-
Plasmid: 10 µl<br>
+
MgCl2: 5µl<br>
-
water: 12.5 µl<br>
+
water: 33µl<br>
 +
DNA (425 or 428): 5µl<br>
 +
 
 +
- PCR:<br>
 +
DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 (0,3µl)<br>
 +
primers (#95, #7): 1,5µl<br>
 +
high fidelity buffer: 5µl<br>
 +
dNTPs: 1µl<br>
 +
TMenzyme: 0,3µl<br>
 +
water: 39,7µl<br>
 +
<br>* Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
 +
<br>*analysis of sequences from 28.09.09
 +
<br>*digest of pExStrepDigSplitFoka prep from 30.09.09
 +
Plasmid: 15 µl<br>
 +
water: 9 µl<br>
BSA: 0.5 µl NEB iGEM stock<br>
BSA: 0.5 µl NEB iGEM stock<br>
-
buffer: 3 µl buffer 1 NEB KuKlabstock<br>
+
buffer: 3 µl buffer 3 NEB iGEM stock<br>
-
Restriction_enzyme_1 : 1 µl AgeI from NEB KuKlabstock<br>
+
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
-
Restriction_enzyme_2 : 2 µl PstI from NEB KuKlabstock <br>
+
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
-
<br>*Gel extraction of pEX_His_FluA_Split_Foki_36GSli (prep 02.09.09 rest4 tube3)<br>
+
<br>*digest of PCR product RBSStrepDigSplitFoka from 30.09.09
-
<br>*Ligation of vector:pEX_His_FluA_Split_Foki_36GSli(prep 02.09.09 rest4 tube3) with insert:Foka(Gelex 17.08)<br>
+
Plasmid: 10 µl <br>
-
Vector: 3 µl pEX_His_FluA_Split_Foki_36GSli from 02.09.09<br>
+
water: 14 µl<br>
-
Insert: 6 µl Foka(Gelex 17.08)<br>
+
BSA: 0.5 µl NEB iGEM stock<br>
-
buffer: 10 µl Quick ligase buffer from NEB iGEM stock<br>
+
buffer: 3 µl buffer 3 NEB iGEM stock<br>
-
Ligase: 1 µl Quick ligase NEB iGEM stock<br>
+
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
-
<br>*Conzentrated AGO 5x via Vivaspin to final conzentration of 3µM (2ml)
+
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
-
<br>*Exchanged buffer of 900µl of Aa AGO solution via dialysis
+
<br>*digest of pExHisFluaSplitFoki prep from 24.08.09
-
<br>*Started PCR procedure to create Aa AGO as an BB part via megaprimer technic
+
Plasmid: 15 µl <br>
-
<br>*His-Dig purification of protein expression from 02.09.09 (pExHisDigSplitFoka)
+
water: 9 µl<br>
-
<br>*SDS gel of His-Dig purification HisDigSplitFoka
+
BSA: 0.5 µl NEB iGEM stock<br>
-
[[Image:Freiburg09_040909_pg_HisDigSplitFoka_vom030909015.jpg|none|thumb|SDS-Gel; pExHisDigSplitFoka ; Lanes: NEB prestained protein marker, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 5, Elution fraction 6, flow through fraction 1, flow through fraction 3, washing fraction 1|400x400px]]
+
buffer: 3 µl buffer 2 NEB iGEM stock<br>
-
<br>*changed dialysis buffer, again with 200 ml (10:00 o'clock)
+
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock<br>
 +
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
 +
-> put in 37°C room for 2h
 +
->preparative gel<br>
 +
[[Image:Freiburg09_011009_fokverdaue.JPG|none|thumb|Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka ||  400x400px]]
 +
interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration<br>
 +
->gelextraction <br>
 +
ligation of<br>
 +
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)<br>
 +
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)<br>
 +
<br>*Inoculation of
 +
- pJS419_StrepDigSplitFoka<br>
 +
- pJS419_HisDigSplitFoka<br>
 +
- pEx_HisFluA<br>
 +
- pEx_HisDigMiddleLiFoka<br>
 +
<br>*digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
 +
<br>*Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09
 +
--> make glycerol stocks tomorrow
 +
<br>*agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
 +
<br>*cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more)
 +
<br>*gel slices stored at -4°C over night
-
<h3>04.09.09, Manu, Hannes, Christoph, Laura, Sarah,Timo</h3>
+
<h3>02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max</h3>
-
<br>*Ordered additional Oligo for M13mp18 cleavage with AGO: "Oligo A4" AAGTTTTTTGGGGTCGAGGTG should allow the Aa Argonaute protein to cut the M13mp18 ssDNA behind base 100. combination with A1 (cuts at 3100) should result in two destinc bandes on the gel with  a 1:2 size ratio
+
<br>* phage display
-
<br>*AGO: over night dialysis to exchange buffer to the "AGO assaybuffer". NanoDrop: 0.575 mg/ml --> 6µM in ca. 1.2 ml buffer -> -20°C freezer
+
- OD600 of the pre-culture should be 0,2 <br>
-
<br>*AGO cleavage assay with M13mp18 ssDNA was roughly performed following the original RNA assay (Ma et al.): <br>
+
- 37°C shaker<br>
-
15µl of the Aa Argonaute protein in the "Ago assay buffer" were incubated 30 minutes with 1µl of 100µM of the guideoligo A1 (ACAACCATCGCCCACGCATAA)at 55°C <br>
+
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)<br>
-
Then 20µl of M13mp18 ssDNA (248ng/µl)was added and incubated for 30 minutes at 55°C<br>
+
- Apportion culture into 10x50 ml Falkon tubes<br>
-
One half of the solvent was treated with proteinase K solution, the other was untreated added to the agarose gele
+
- 15 minutes on ice<br>
-
--> we obtained 2 bands which might be the circular M13 DNA and the linear M13 DNA
+
- Centrifuge 10 minutes at 4°C / 2500 g<br>
-
[[Image:Freiburg 09 AGO M13 verdau 45min.JPG|none|thumb|Agarose gele of AGO M13 digest; Lanes: Marker:1kb ladder, AGO M13 digest, AGO M13 digest treated with Proteinase K, untreated M13 ssDNA|400x400px]] <br>
+
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)<br>
-
On lane two there is a clearly visible bandshift. The band on the very bottom of lane three is most likely the guide oligo that was released when the AGO was destroyed by the proteinase.<br>
+
- resuspend pellets in 25 ml H2O (10-> 8 tubes)<br>
-
Next week we will do another assay with two guide oligos (A1 and A4) that should result in two distinct bands and thereby allow as to exclude the possibility that the bandeshift is due to AGO bound to the targed ssDNA
+
- 15 minutes on ice<br>
-
<br>*AGO PCR to craeat an Aa AGO BB part failed and will be repeated on Monday
+
- Centrifuge 10 minutes at 4°C / 2500 g<br>
 +
- Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 tubes)<br>
 +
- 15 minutes on ice<br>
 +
- Centrifuge 10 min at 4°C / 2500 <br>
 +
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> 2 tubes)<br>
 +
- 5 minutes on ice<br>
 +
- Centrifuge 10 minutes at 4°C / 2500 g<br>
 +
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)<br>
 +
- 5 minutes on ice<br>
 +
- Centrifuge 10 min at 4°C / 2500 g<br>
 +
- Discard supernatant, pool pellets in 1 ml 10% DMSO<br>
 +
- Measure OD600 (1:100 dilution), OD should be 0,4<br>
 +
- Make aliquots à 80 µl, souse with N2<br>
 +
- Store at -80°C<br>
 +
<br>*glycerol stock of pEx_HisFluASplitFoki in BL21de3
 +
<br>*SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09
 +
--> do a Western Blot
-
<h3>06.09.09, Hannes</h3>
+
<br>*new ampicilin 100 aliquots
-
<br>*Inoculation of:
+
<br>*preparation of M13dsDNA
-
- BL21de3 pEx-StrepDigSplitFoka<br>
+
<br>*chem. competent cells aliqots
-
- BL21de3 pEx-HisDigSplitFoka<br>
+
-
- pEx-HisFluASplitFoki-36GS-Foka<br>
+
-
<h3>07.09.09, Laura, Manu, Gerrit, Christoph, Isabel, Anika</h3>
 
-
<br>*Prepared LB Medium (10l)<br>
 
-
<br>*Purification via Strep-Tactin column of Strep-Dig-Split-Foka
 
-
<br>*SDS Gel with eluate fractions E1 til E7 and washfractions W2 til W3 and NEB prestained proteinmarker
 
-
---> see picture
 
-
result: no fusioned protein StrepDigSplitFoka eluated
 
-
<br>*Plamidpreparation of pEx His-FluA-Split-Foki-36GSLi-Foka, glycerol stocks and sequencing
 
-
<br>*Glycerol stocks of pEx-Strep-Dig-Split-Foka and pEx-His-Dig-Split-Foka
 
-
<br>*Cutting experiments and analytical agarose gel of digested M13 DNA hybridized with Fok control 1 and 2, pure oligos 1 and 2, pure ds and ss M13 DNA, 1kb ladder
 
-
[[Image:2009-09-07_M13_2.Bild.jpg |none|thumb|Analytical Agarose Gel of M13 Digest; Lanes: Marker:100bp ladder, Fok Control Oligo 1, Fok Control Oligo 2, M13 ssDNA Hybridized with Fok Control 1 and 2, M13 ssDNA Hybridized with Fok Control 1 and 2, ds and ss M13 DNA|400x400px]]
 
-
<br>*Starter culture of pEx-His-Flua-Split-Foki(amp plate of 25.08.09)in BL21DE3 (200 ml LB + amp)
 
-
<br>*Hybridisation of long, middle and short linker
 
-
<br>*pEx His-FluA-Split-Foki-36GSLi-Foka is now called <b>FokM</b> [http://www.molbiotech.uni-freiburg.de/iGEM/wiki2009/index.php/Fok_monomer Sequenz]
 
-
<br>*Transformation of FokM into BL21 de3 gold
 
-
<h3>08.09.09, Manu, Gerrit, Christoph, Sarah, Anika, Hannes, Laura, Isabel, Julia</h3>
+
<br>*Periplasma Project
-
<br>*made LB Agar
+
-
<br>*poured amp plates (3 plates left)
+
-
<br>*ligation of short, middle and long linker (hybridized at 07.09.09) and pMA vector (gelex from 27.08.09)
+
-
<br>*ligation of short, middle and long linker (hybridized at 07.09.09) and pMA_Foki (gelex from 28.08.09) and pMA_Foka (gelex from 27.08.09)
+
-
<br>*made SDS gel of collected pellets (T0-3) from expression of StrepDigSplitFoka and HisDigSplitFoka
+
-
aim: to see if induction of expression of fusioned protein was successful
+
-
[[Image:Freiburg09_090909_pg_HisDigSplitFoka_SDS017.jpg|none|thumb|SDS-Gel; pExHisDigSplitFoka ; Lanes: NEB prestained protein marker, expression fraction T0, T1, T2, T3, T1, T2, T3|400x400px]]
+
-
[[Image:Freiburg09_090909_pg_StrepDigSplitFoka_SDS016.jpg|none|thumb|SDS-Gel; pExStrepDigSplitFoka ; Lanes: NEB prestained protein marker, expression fraction T0, T1, T2, T3, T1, T2, T3|400x400px]]
+
-
<br>*Stripping of Ni-NTA column
+
-
<br>*made starter culture (300ml LB+Amp+1%Glucose) with pEXHisDigSplitFoki in BL21de3
+
-
<br>*Made gradient PCR (+-5 in first 10 cycles; +-2.5 in last 14 cycles) to generate BB Aa AGO part. Therefor optimised pcr protocoll and made an additional approach using DMSO
+
-
<h3>09.09.09, Gerrit, Christoph, Sarah, Isabel, Anika, Hannes, Laura, Max, Julia, Dieter</h3>
+
Digestion:<br>
-
<br>*expression of pExHisFluASplitFoki in 6 times 1 ml LB and Amp
+
-
- pellets were taken at T0 (time of induction with IPTG), T1, T2, T3, T4<br>
+
-
- at the end culture was centrifuged down at 4000rpm, 20 min, 4°C<br>
+
-
- pellets frozen in -80° C<br>
+
-
<br>* Repetition of AGO cleavage assay, using oligos A1 and/or A4 to determine whether bandshift was due to AGO bound to the DNA or cleavege of the ssDNA target-->  Agarose gel revealed several promising bands (see picture). Results have to be dicussed yet.
+
-
<br>* test digestion of FokM shows that vector and insert are to small
+
-
<br>* test expression of FokM aborted
+
-
<br>* new cloning of FokM has to be done tomorrow
+
-
<br>*new measurement of concentration of HisFluASplitFoki (dialysed at 03.09.09)
+
-
<br>*Agarosegel of yesterdays gradient PCR again showed none of the expected bands.<br>
+
-
-> Gel of the used template "Aa AGO in pET28a 27.08.09 1" revealed an unexpected strong band at app. 20kb. pET28a with the Aa AGO as an insert should be 7.5kb in size. No such band was observed.
+
-
<br>* Therefore a new PCR was started using the original optimised PCR protocol (first annealing temperature 55°C, second 65°C) using the sample "Aa AGO in pET28a 27.08.09 2" as a template
+
-
<br>*measurement of fluorescence of FluA marked oligo, protein, and oligo and protein (quenching)
+
-
<br>*inoculation of short, middle and long linker in pMA, pMAFoki and pMAFoka
+
-
<h3>10.09.09, Manu, Hannes, Christoph, Timo, Gerrit</h3>
+
- digested with xba and spe
-
<br>*His-tag purification with Ni-NTA column of expression products from yesterday (09.09.09)- HisFluASplitFoki
+
  --> GelBilder
-
<br>*Glycerin Stocks of:
+
-
pMA short linker 1,2 + Fok a/i 1,2 from 09.09.09<br> 
+
-
pMA middle linker 4,5 + Fok a/i 4,5 from 09.09.09<br>
+
-
pMA long linker 1,2 + Fok a/i 1,2 from 09.09.09<br>  
+
-
<br>*Pellets of: 
+
-
pMA short linker 3,4,5,6 + Fok a/i 3,4,5,6 from 09.09.09<br> 
+
-
pMA middle linker 1,2,3,6 + Fok a/i 1,2,3,6 from 09.09.09<br>
+
-
pMA long linker 3,4,5,6 + Fok a/i 3,4,5,6 from 09.09.09<br>
+
-
<br>*digestion of pEX_His_fluA_Split_Foki of 26.08.09 with NgoMIV and PstI
+
-
<br>*digestion of pMA_36GSlinker of 28.08.09 AgeI and PstI
+
-
<br>*gelextraction of vector and insert
+
-
<br>*quick ligation of pEX_His_FluA_Split_FokI and 36GSLi
+
-
<br>*transformation of pEX_His_FluA_Split_FokI+36GSLi into RV308
+
-
<br>*plasmid preparation of pMA short linker, pMA middle linker, pMA long linker
+
-
<br>*plasmid preparation of pEX-short/middle/long-Linker-Fok(active/inactive)
+
-
<br>*digestion of pEX-short/middle/long-Linker-Fok(active/inactive) with NgoMIV and PstI, over night at 37°C
+
-
'''Nanodrop-Data:'''
+
<br>* Gelextraction
-
<table border=1 cellpadding=0 cellspacing=0 width=1055 style='border-collapse:
+
Min Elute Gel Extraction Kit<br>
-
collapse;table-layout:fixed;width:791pt'>
+
- mesure mass: StrepFokA = 50 mg<be>
-
<tr height=20 style='height:15.0pt'>
+
- JS418= 90mg<br>
-
  <td height=20 class=xl6323721 width=260 style='height:15.0pt;width:195pt'>Sample
+
-JS419=80mg<br>
-
  ID</td>
+
- HisFokA = 250mg<br>
-
  <td class=xl6423721 width=72 style='border-left:none;width:54pt'>User ID</td>
+
-
  <td class=xl6423721 width=83 style='border-left:none;width:62pt'>Date<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>Time<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>ng/ul<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>A260<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>A280<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>260/280<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>260/230<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>Constant<span
+
-
  style='mso-spacerun:yes'> </span></td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>Cursor Pos.</td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>Cursor abs.</td>
+
-
  <td class=xl6423721 width=64 style='border-left:none;width:48pt'>340 raw</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:38</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>140,47</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,809</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,461</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,92</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,12</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,328</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,031</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:39</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>117,82</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,356</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,234</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,91</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,99</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,186</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,027</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:40</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>131,21</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,624</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,349</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,94</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,18</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,203</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,019</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:40</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>158,86</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,177</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,668</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,9</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,85</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,719</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,07</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:41</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>197,16</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,943</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,059</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,92</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,06</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,911</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,032</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:42</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>137,14</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,743</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,427</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,92</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,19</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,251</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,03</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker-Foka
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:43</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>173,32</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,466</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,8</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,93</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,25</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,543</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,03</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker-Foka
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:44</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>151,38</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,028</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,571</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,93</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,24</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,353</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,021</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker-Foki
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:44</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>156</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,12</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,639</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,9</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,21</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,413</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,021</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Long_Linker-Foki
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:45</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>167,47</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,349</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,735</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,93</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,22</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,511</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,023</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker-Foki
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:45</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>160,29</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,206</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,68</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,91</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,21</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,449</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,017</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker-Foki
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:46</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>159,35</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,187</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,648</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,93</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,23</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,429</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,022</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker-Foka
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:47</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>207,54</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>4,151</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,191</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,89</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,21</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,876</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,02</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Short_Linker-Foka
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:47</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>200,95</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>4,019</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,107</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,91</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,21</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,82</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,02</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker-Foki
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:48</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>185,79</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,716</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,939</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,92</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,22</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,671</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,01</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker-Foki
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:49</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>180,42</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,608</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,865</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,93</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,22</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,622</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,016</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker-Foka
+
-
  1</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>196,37</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,927</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,997</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,97</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,19</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,796</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,022</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pMA-Middle_Linker-Foka
+
-
  2</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>12:50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>180,62</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>3,612</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,895</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,91</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,25</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,606</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>-0,011</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>NgoMIV_36GSLi_PstI</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>15:40</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>1,72</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,034</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>-336,01</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,01</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>5,671</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0</td>
+
-
</tr>
+
-
<tr height=20 style='height:15.0pt'>
+
-
  <td height=20 class=xl6323721 style='height:15.0pt;border-top:none'>pEX_His_FluA-Split_FokI_AgeI_PstI</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>FreiGEM</td>
+
-
  <td class=xl6523721 style='border-top:none;border-left:none'>10.09.2009</td>
+
-
  <td class=xl6623721 style='border-top:none;border-left:none'>15:41</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,52</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0,01</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>-0,017</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>-0,6</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>0</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>50</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>230</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>2,615</td>
+
-
  <td class=xl6423721 style='border-top:none;border-left:none'>-0,01</td>
+
-
</tr>
+
-
  <tr height=0 style='display:none'>
+
-
  <td width=260 style='width:195pt'></td>
+
-
  <td width=72 style='width:54pt'></td>
+
-
  <td width=83 style='width:62pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
  <td width=64 style='width:48pt'></td>
+
-
</tr>
+
-
</table>
+
-
<h3>11.09.09, Manu, Hannes, Christoph, Laura, Anika</h3>
+
Nandropdata<br>
-
<br>*labtalk
+
-
<br>*SDS gel of protein purification from yesterday (10.09.09) HisFluASplitFoki
+
-
[[Image:Freiburg09 090911 His aufreinigungGel1.jpg|none|thumb|SDS-Gel; pExHisFluASplitFoki ; Lanes: NEB prestained protein marker, elution fractions E1-5, W1-2, D2-3|400x400px]]
+
-
<br>*SDS gel of bacteria pellets during expression of HisFluASplitFoki
+
-
[[Image:Freiburg09_090911_His_aufreinigungGel2.jpg|none|thumb|SDS-Gel; pExHisFluASplitFoki ; Lanes: NEB prestained protein marker, expression fraction T0, T1, T2, T4, T5|400x400px]]
+
-
[[Image:Freiburg09_P7708S_prestained_proteinmarker_broad_range.gif|none|thumb|NEB prestained protein marker broad range|400x400px]]
+
-
<br>*concentrations of eluate fractions 1-4 weren't measureable at photometer and nanodrop (weird spectra)
+
<br>*Ligation
-
<br>*Eluate fractions 1-4 were frozen at -80°C
+
- with Quickligase
-
<br>*Checking of the plates pEX_His_FluA_Split_Foki_36GsLi RV308 (Trafo of 10.09.09) showed none Bacteriagrowth, perhaps problems with the GelEX
+
-
<br>*gelextraction of NgoMIV-Short/Middle/LongLinker-FokA-PstI (the Fok(inactive) digests didn't work: no bands on the gel)<br><br>
+
-
<h3>14.09.09, Gerrit, Manu, Laura, Anika, Isabel, Timo </h3>
+
JS119-StrepFokA<br>
-
<br>*fetched a new QuickLigase-Kit at the NEB-Freezer
+
JS118-Strep<br>
-
<br>*aliquoted the QuickLigase Buffer to 20 µl aliquots
+
JS118-HisFokA<br>
-
<br>*Ligation of vector:pEX_His_FluA (gelex from 20.08.09) with insert: Short/Middle/Long-Linker-Foka(Gelex from 11.09.09)<br>
+
JS19-HisFokA<br>
-
Vector: 3 µl pEX_His_FluA from 20.08.09 (digested with AgeI and PstI)<br>
+
-
Insert: 6 µl Short/Middle/Long-Linker(Gelex from 11.09.09, digested with NgoMIV and PstI)<br>
+
-
buffer: 10 µl Quick ligase buffer from NEB iGEM stock<br>
+
-
Ligase: 1 µl Quick ligase NEB iGEM stock<br>
+
-
<br>*Ligation of vector:pEX_His_Dig (gelex from 27.08.09) with insert: Short/Middle/Long-Linker-Foka(Gelex from 11.09.09)<br>
+
- 15 min at 25°C
-
Vector: 3 µl pEX_His_FluA from 27.08.09 (digested with AgeI and PstI)<br>
+
-
Insert: 6 µl Short/Middle/Long-Linker(Gelex from 11.09.09, digested with NgoMIV and PstI)<br>
+
-
buffer: 10 µl Quick ligase buffer from NEB iGEM stock<br>
+
-
Ligase: 1 µl Quick ligase NEB iGEM stock<br>
+
-
<br>*Ligation of vector:pEX_Strep_Dig (gelex from 27.08.09) with insert: Short/Middle/Long-Linker-Foka(Gelex from 11.09.09)<br>
+
<br>*Transformation
-
Vector: 3 µl pEX_His_FluA from 27.08.09 (digested with AgeI and PstI)<br>
+
- 2YT-Medium 950 µL to 50µl cells and DNA <br>
-
Insert: 6 µl Short/Middle/Long-Linker(Gelex from 11.09.09, digested with NgoMIV and PstI)<br>
+
-
buffer: 10 µl Quick ligase buffer from NEB iGEM stock<br>
+
-
Ligase: 1 µl Quick ligase NEB iGEM stock<br>
+
-
<br>*Ligation of vector:pMA_Foki (gelex from 27.08.09) with insert: Short/Middle/Long-Linker(hybridized 07.09.09)<br>
+
BL21-JS190<br>
-
Vector: 3 µl pMA_Foki (digested with XbaI and NgoMIV)<br>
+
BL21-JS119StrepFokA<br>
-
Insert: 6 µl Short/Middle/Long-Linker(cutting sites: NgoMIV and PstI)<br>
+
BL21-JS118Strep<br>
-
buffer: 10 µl Quick ligase buffer from NEB iGEM stock<br>
+
BL21-JS118HisFokA<br>
-
Ligase: 1 µl Quick ligase NEB iGEM stock<br>
+
BL21-JS19HisFokA
-
<br>*transformation of pEx-His-FluA-Short/Middle/LongLinker-Fok(active) into RV308
+
-
<br>*transformation of pEx-His-Dig-Short/Middle/LongLinker-Fok(active) into RV308
+
-
<br>*transformation of pEx-Strep-Dig-Short/Middle/LongLinker-Fok(active) into RV308
+
-
<br>*transformation of pMA-Short/Middle/LongLinker-Fok(inactive) into RV308
+
-
<br>*prepared SDS gels
+
-
<br>*Digest of pMA_Foki(prep I 05.08.09) with XbaI and NgoMIV<br>
+
-
Plasmid: 10 µl<br>
+
-
water: 14.5 µl<br>
+
-
BSA: 0.5 µl NEB iGEM stock<br>
+
-
buffer: 3 µl buffer 4 igem stock<br>
+
-
Restriction_enzyme_1 : 1 µl XbaI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1 µl NgoMIV from NEB KuKlabstock <br>
+
-
-> preparative gel, freezed gel slice in case ligation won't work again
+
-
<br>*test-digest of pMA_short/middle/longlinker_Foka(prep 10.09.09) with FokI<br>
+
-
Plasmid: 4 µl<br>
+
-
buffer: 0.5 µl buffer 4 igem stock<br>
+
-
Restriction_enzyme_1 : 0.5 µl FokI from igem stock<br>
+
-
-> analytical gel
+
-
<br>*ordering of fluorscin marked oligos
+
-
<br>*preparation of competent cells: RV308, there is no antibiotic in the approach because this bacterial strain has no resistance(on shaker in 37°C romm over night until tomorrow)
+
-
<br>*planning of Fos_bZip with freigem restricion sites
+
-
<br>* Sequencing of
+
- Plated on KM plats<br>
-
- pMAShortlinker clone 1 plasmid prep of 10.09
+
<br>* Preparation of overnight cultures 5 ml LB medium each
-
- pMAmiddellinker clone 2(4) plasmid prep of 10.09
+
- pBad+Kan<br>
-
- pMAlonglinker clone 1 plasmid prep of 10.09
+
-pET39B(+)<br>*Amp<br>
-
- pMAShortlinker+FokA clone 2 plasmid prep of 10.09
+
--> Stored in 37°C room
-
- pMAShortlinker+FokI clone 2 plasmid prep of 10.09
+
-
- pMAmiddellinker+FokA clone 4(2) plasmid prep of 10.09
+
-
- pMAmiddellinker+FokI clone 4 plasmid prep of 10.09
+
-
- pMAlonglinker+FokI clone 1 plasmid prep of 10.09
+
-
- pMAlonglinker+FokA clone 1 plasmid prep of 10.09
+
-
<br>* digest with HaeII (+76, NEB IV, BSA) of 28 minipreps
+
<br>*Digest:
-
Plasmid: 5 µl<br>
+
- pMASplitFoka<br>
-
water: 3,6 µl<br>
+
- pMAFoka<br>
-
BSA: 0.1 µl (100x)from iGEM stock<br>
+
- pMAFoki<br>
-
buffer NEB IV: 1 µl buffer (10x)from iGEM stock<br>
+
- pMASplitFoki<br>
-
Restriction_enzyme_1 : 0,3 µl HaeII<br>
+
- pMALongLiFoka<br>
 +
- pMALongliFoki<br>
 +
- pMAShortli<br>
 +
- pMAMiddleli<br>
 +
- pMALongli<br>
 +
- pExStrepFluA<br>
-
<br>*preparative gel (1%)
 
-
Marker 1kb; -Eco 86/88; -Eco 86/89; TTT 86/88; TTT 86/89; Taq 86/88; Taq 86/89
 
-
Marker 1kb; -Eco 87/88; -Eco 87/89; TTT 87/88; TTT 87/89; Taq 87/88
 
-
<br>*gel extraction of (kit from Janina)
+
<br>*preparative gel
-
Taq 86/88, Taq 86/89, Taq 87/88, Taq 87/89 in 50 µl 70°C water
+
[[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA<|400x400px]]
 +
->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly
 +
<br>*gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09
-
<br>*analytic gel (1%)
 
-
Marker 1kb; samples 442-1, 442-2, 442-3; samples 443-1, 443-2, 443-3; 427-1 to -12; 424-1 to -12; Taq 87/89
 
-
<br>*transformation of EcoRI-removal
+
<br>* digest and ligation into new pMA:<br>
-
ligation TorA-flag and DsbA-flag, vector control
+
-pMASplitFokA clone1 19.08.09<br>
 +
-pMAFokA clone2 05.08.09<br>
 +
-pMAFoki clone2 05.08.09<br>
 +
-pMASplitFoki clone2 06.08.09<br>
 +
-pMAshortLi clone2 10.09.09<br>
 +
-pMAmiddelLi clone2 10.09.09<br>
 +
-pMAlongLi clone1 10.09.09<br>
 +
-pMAlongLiFokA clone1 10.09.09<br>
 +
-pMAlongLiFoki clone1 10.09.09<br>
 +
digestion with EcoRI and SpeI (vector and insert)
 +
<br>
 +
<br>* Transformation of:<br>
 +
-pMAFokA clone2 05.08.09<br>
 +
-pMAFoki clone2 05.08.09<br>
 +
-pMASplitFoki clone2 06.08.09<br>
-
<br>* Inoculation of:
+
<br>*plasmidprep of:<br>
-
- startingculture His+Dig+Split+FokA (from 31.08.09)
+
1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br>
-
200ml LB+200µl Amp + 2g glucose --> 24°C 180rpm
+
2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br>
 +
3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br>
-
<h3>15.09.09, Manu, Hannes, Gerrit, Anika, Sarah, Laura </h3>
+
4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br>
-
<br>*over-night culture of pEX-His-Dig-Split-Fok(active) showed an OD(600) of 0,132. No expression was started.
+
5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br>
-
<br>*the LB/Amp plates of pEx-His/Strep-Flua/Dig-Short/Middle/LongLinker-Fok(active) show only few colonies. One construct is completly missing (pEx-His-Dig-ShortLinker-Fok(active)).
+
6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br>
-
<br>*the LB/Amp plates of pMA-Short/Middle/LongLinker-Fok(inactive) showed many colonies.
+
-
<br>* Plamidpreparation of RV308 pMA_36GSLi Klon3/4(plate from 27.08.09), glycerol stocks and sequencing
+
-
<br>* continued phage display:
+
-
digestion of 442-1 of 14.09.09 with XmnI and NheI<br>
+
-
Plasmid: 20 µl<br>
+
-
water: 3,7 µl<br>
+
-
BSA: 0.3 µl (100x)from iGEM stock<br>
+
-
buffer NEB IV: 3 µl buffer (10x)from iGEM stock<br>
+
-
Restriction_enzyme_1 : 1,5 µl XmnI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1,5 µl NheI from NEB KuKlabstock<br>
+
-
digestion of 443-1 of 14.09.09 with XmnI and NheI<br>
+
-
Plasmid: 18 µl<br>
+
-
water: 5,7 µl<br>
+
-
BSA: 0.3 µl (100x)from iGEM stock<br>
+
-
buffer NEB IV: 3 µl buffer (10x)from iGEM stock<br>
+
-
Restriction_enzyme_1 : 1,5 µl XmnI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1,5 µl NheI from NEB KuKlabstock<br>
+
-
finished transformation of 436;423-427 and V/1;V/6;V/411;V/375<br>
+
-
<br>*chemical competent cells of RV308
+
-
<br>*glycerol stock of RV308
+
-
<br>*preparation of electrical competent cells: RV308, there is no antibiotic in the approach because this bacterial strain has no resistance(on shaker in 37°C romm over night until tomorrow)
+
-
<br>*simulated Hybridization of M13 DNA - hybridization with Fok control 2 & 3 oligos<br>
+
-
- 10µl M12 ssDNA<br>
+
-
- 10µl MgCl from iGEm Stock<br>
+
-
- 5µl Tris-HCl<br>
+
-
- 2µl Fok-Kontrolle 2 (org. Tube from 28.07.09)<br>
+
-
- 2µl Fok-Kontrolle 3 (org tube from 06.08.09)<br>
+
-
<br>* Testdigest with FokI with:<br>
+
-
10µl M13DNA+Oligo2+3<br>
+
-
1,2µl Puffer4<br>
+
-
1µl FokI<br>
+
-
Gel:<br>
+
7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit1<br>
-
Marker / ssDNA+Oligo2+3+Fok / ssDNA+Oligo2+3+Fok / ssDNA+Oligo2+3 37° / ssDNA+ Oligo2+3 4°<br>
+
8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit2<br>
-
BILD<br>
+
9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br>
-
<br>*ordered His_GSG_Fos_bZip
+
-
<h3>16.09.09,Gerrit, Anika, Sarah, Laura, Isabel </h3>
+
10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit3<br>
 +
11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit4<br>
 +
12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br>
-
<br>*made plasmid preparation of
+
<br>*testdigest of:
-
pEX-Strep-Dig-middleLinker-FokA clone 1 and clone 2<br>
+
V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick<br>
-
pEX-Strep-Dig-shortLinker-FokA clone 1 and clone 2<br>
+
V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick<br>
-
pEX-His-Dig-middleLinker-FokA clone 1 and clone 2<br>
+
V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick<br><br>
-
pEX-His-FluA-shortLinker-FokA clone 1 and clone 2<br>
+
-
pEX-His-FluA-middleLinker-FokA clone 1 and clone 2<br>
+
-
pMA-shortLinker-FokI clone 1 and clone 2<br>
+
-
pMA-middleLinker-FokI clone 1 and clone 2<br>
+
-
pMA-longLinker-FokI clone 1 and clone 2<br>
+
-
glycerol stocks stored in "iGEM AA" at -80°C
+
V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick <br>
 +
V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick<br>
 +
V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick<br><br>
-
<br>*continued phage display
+
V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 <br>
-
- plasmid preparation of:<br>
+
V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 <br>
-
444-1,444-2,444-3;445-1,445-2,445-3;443-1;442-1<br>
+
V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3<br><br>
-
- sequencing of 444-1 and 445-1 with primer:sfLacP1<br>
+
-
- digest of 444-1 and 445-1 with SfiI and NheI<br>
+
-
'''444-1'''<br>
+
-
Plasmid: 19 µl<br>
+
-
water: 5 µl<br>
+
-
BSA: 0.3 µl (100x)from iGEM stock<br>
+
-
buffer NEB IV: 3 µl buffer (10x)from iGEM stock<br>
+
-
Restriction_enzyme_1 : 1,5 µl SfiI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1,5 µl NheI from NEB KuKlabstock<br>
+
-
'''445-1'''<br>
+
-
Plasmid: 15 µl<br>
+
-
water: 9 µl<br>
+
-
BSA: 0.3 µl (100x)from iGEM stock<br>
+
-
buffer NEB IV: 3 µl buffer (10x)from iGEM stock<br>
+
-
Restriction_enzyme_1 : 1,5 µl SfiI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1,5 µl NheI from NEB KuKlabstock<br>
+
-
- preparative gel (1%)of digest of 442-1 and 443-1, 440-5<br>
+
-
<br>*expression cultures (4 flasks à 1l) of HisDigSplitFoka
+
-
- took pellets at T0-T4<br>
+
-
- centrifuged at 4000 rpm, 4°C, 20 min<br>
+
-
- froozen pellets in -80°C<br>
+
-
<br>*made 5l LB medium
+
-
<br>* electrical competent cells of RV308, stored in box at -80°C
+
-
<h3>17.09.09,Hannes, Anika, Sarah, Laura, Isabel, Timo, Manuel </h3>
+
V10. pEX_DsbA+His+Dig+Split+FokA Clone1 <br>
-
<br>*Digest of pEX_His_FluA (prep 20.08.09 clone), pExHisDig (prep 27.08.09, clone 1) and  pExStrepDig (prep 27.08.09, clone 1) with AgeI and SpeI<br>
+
V11. pEX_DsbA+His+Dig+Split+FokA Clone2 <br>
-
Plasmid: 10 µl<br>
+
V12. pEX_DsbA+His+Dig+Split+FokA Clone3<br><br>
-
water: 14.5 µl<br>
+
-
BSA: 0.5 µl NEB iGEM stock<br>
+
-
buffer: 3 µl buffer 4 NEB iGEM stock<br>
+
-
Restriction_enzyme_1 : 2 µl AgeI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
+
-
<br>*Digest of pMA_Shortli_Foki (prep 10.09.09 clone 2), pMA_Middleli_Foki (prep 10.09.09 clone 4) and  pMA_Longli_Foki (prep 10.09.09 clone 1) with NgoMIV and SpeI<br>
+
-
Plasmid: 10 µl<br>
+
-
water: 14.5 µl<br>
+
-
BSA: 0.5 µl NEB iGEM stock<br>
+
-
buffer: 3 µl buffer 4 NEB iGEM stock<br>
+
-
Restriction_enzyme_1 : 1 µl NgoMIV from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
+
-
-> stored in -20°C
+
-
<br>*preparation of binding buffer, washing buffer I and II and elution buffer for the ni-column
+
-
<br>*purification of the expression cultures of 16.09.09 HisDigSplitFoka  (ni-column)
+
-
<br>*'''continued the phage display:'''
+
-
<br>*preparation of 425-1 to 425-10; 428-1 to 428-10; 423-1,424-1,426-1,427-1,429-1,436-1<br>
+
-
<br>*test digest of all 425 and 428 probes with HaeII<br>
+
-
DNA: 5 µl<br>
+
-
water: 3,7 µl<br>
+
-
BSA: 0.1 µl NEB iGEM stock<br>
+
-
buffer: 1 µl buffer 4 NEB iGEM stock<br>
+
-
Restriction_enzyme : 0,2 µl HaeII from NEB KuKlabstock<br>
+
-
<br>*dephosphorylation of the vector fragments 444 and 445 with SAP to stop the digest (2x1µl for 30min 37°C and 1X20min 70°C)<br>
+
-
<br>*preparative gel of the vector fragments pJs#375(digest and re-digest), 444 and 445 and gel extraction<br>
+
-
<h3>18.09.09 Isabel, Sarah, Laura, Manu, Timo, Hannes, Anika </h3>
+
V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br>
-
'''<br>*continued phage display'''
+
V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)<br>
-
<br>*sequencing of 423,424,426,427,429,436<br>
+
with NcoI-HF and XbaI 1h at 37°C<br>
-
<br>*analytical gel of test digests of 425 (1-10) and 428 (1-10) and sequencing of
+
-
<br>*ligation of V/444, V7445 and V/375 re-digest with 3 approaches:
+
-
'''1)''' 1µl vector<br>
+
-
5µl insert 10<br>
+
-
1µl ligase buffer<br>
+
-
1µl T4 ligase<br>
+
-
2µl water<br>
+
-
'''2)''' 1µl vector<br>
+
-
5µl insert 11<br>
+
-
1µl ligase buffer<br>
+
-
1µl T4 ligase<br>
+
-
2µl water<br>
+
-
'''3)''' 1µl vector<br>
+
-
1µl ligase buffer<br>
+
-
1µl T4 ligase<br>
+
-
7µl water<br>
+
 +
--> geldbild
-
<h3>19.09.09 Julia </h3>
+
<h3>03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph </h3>
 +
<br>*plasmid prep of pET and pBad and Glytsocks(Xbl)
 +
<br>*new aliquots Ampicilin (70%EtOH)
 +
<br>*Test digest M13dsDNA+fokI as control for dsDNA , the pictures showed the two expected lanes(hardly visible on the printout).
-
<br>*Put plates from 37°C and digestion from 37°C room to the cooling room at 4°C
+
[[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel; M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09, M13dsDNA02.10.09 digest fokI |400x400px]]
-
<br>*Centrifuged 427-1; 424-1 down and froze the pellet at -20°C
+
<br>*digest of
 +
- pEx-Strep-Dig-LongLinker-Fok(inactive)<br>
 +
- pEx-Strep-Dig-MiddleLinker-Fok(inactive)<br>
 +
- pEx-Strep-Dig-ShortLinker-Fok(inactive)<br>
 +
- pEx-Strep-Dig-SplitLinker-Fok(inactive)<br>
 +
- pEx-His-Dig-SplitLinker_Fok(inactive)<br>
 +
- pEx-His-Dig-SplitLinker_Fok(active)<br>
 +
- pEx-Strep-Dig-SplitLinker-Fok(active)<br>
 +
- pEx-His-Dig-LongLinker-Fok(inactive)<br>
 +
- pEx-His-FluA-LongLinker-Fok(inactive)<br>
 +
- pEx-His-FluA-MiddleLinker-Fok(inactive)<br>
 +
- pEx-His-FluA-ShortLinker-Fok(inactive)<br>
 +
- pEx-His-FluA-SplitLinker-Fok(inactive)<br>
 +
- pEx-His-Dig<br>
 +
- pMA<br>
 +
with NgoMIV and SpeI
 +
<br>*digest of
 +
- pMA<br>
 +
with XbaI and AgeI
-
<h3>21.09.09 Laura, Gerrit, Isabel, Sarah, Hannes, Anika, Julia </h3>
+
<br>*Ligation of:
-
<br>*continued phage display
+
- Strep-Dig-LongLinker-Fok(inactive)<br>
-
-plasmidpreparation of 424-1,424-2,424-3 and 427-1,427-2,427-3<br>
+
- Strep-Dig-MiddleLinker-Fok(inactive)<br>
-
-digestion of 424-1 with XmnI and NheI (37°C) for 6h<br>
+
- trep-Dig-ShortLinker-Fok(inactive)<br>
-
DNA: 58µl<br>
+
- Strep-Dig-SplitLinker-Fok(inactive)<br>
-
buffer IV:11µl<br>
+
- His-Dig-SplitLinker_Fok(inactive)<br>
-
BSA:1,1µl<br>
+
- His-Dig-SplitLinker_Fok(active)<br>
-
enzyme_1: 1,5µl<br>
+
- Strep-Dig-SplitLinker-Fok(active)<br>
-
enzyme_2: 1,5µl<br>
+
- His-Dig-LongLinker-Fok(inactive)<br>
-
-new digest of 444-1 and 445-1 with SfiI (50°C)for 6h and then with NheI (37°C over night)<br>
+
- His-FluA-LongLinker-Fok(inactive)<br>
-
'''444-1'''
+
- His-FluA-MiddleLinker-Fok(inactive)<br>
-
DNA: 19µl<br>
+
- His-FluA-ShortLinker-Fok(inactive)<br>
-
buffer IV:3µl<br>
+
- His-FluA-SplitLinker-Fok(inactive)<br>
-
BSA:0,3µl<br>
+
into pMA<br>
-
water:4,7µl<br>
+
-
enzyme_1: 1,5µl<br>
+
-
enzyme_2: 1,5µl<br>
+
-
'''445-1'''
+
-
DNA: 15µl<br>
+
-
buffer IV:3µl<br>
+
-
BSA:0,3µl<br>
+
-
water:8,7µl<br>
+
-
enzyme_1: 1,5µl<br>
+
-
enzyme_2: 1,5µl<br>
+
-
-ligation of digests of 444-1,445-1 and 375(re-digest)
+
-
<br>*SDS gel of HisDigSplitFoka
+
-
[[Image:Freiburg09_210909_pg_HisDigSplitFoka022.jpg|none|thumb|SDS-Gel; HisDigSplitFoka; Lanes: NEB prestained protein markerbroad range, elution fraction 1-9|400x400px]]
+
-
[[Image:Freiburg09_210909_pg_HisDigSplitFoka_pellets_u_wasch023.jpg|none|thumb|SDS-Gel; HisDigSplitFoka; Lanes: NEB prestained protein markerbroad range, t0 (before induction), t1 (1 hour after induction), t2 (2 hours after induction), t3 (3 hours after induction), t4 (4 hours after induction), flow through fraction 1, flow through fraction 3, washing fraction 1, washing fraction 3|400x400px]]
+
and<br>
-
[[Image:Freiburg09_P7708S_prestained_proteinmarker_broad_range.gif|none|thumb|NEB prestained protein marker broad range|400x400px]]
+
- Strep-Dig-LongLinker-Fok(inactive)<br>
 +
- Strep-Dig-MiddleLinker-Fok(inactive)<br>
 +
- trep-Dig-ShortLinker-Fok(inactive)<br>
 +
into new pEX
 +
<br>*inoculation of:
 +
-pMASplitFokA clone1 19.08.09<br>
 +
-pMAFokA clone2 05.08.09 (new)<br>
 +
-pMAFoki clone2 05.08.09 (new)<br>
 +
-pMASplitFoki clone2 06.08.09 (new)<br>
 +
-pMAshortLi clone2 10.09.09<br>
 +
-pMAmiddelLi clone2 10.09.09<br>
 +
-pMAlongLi clone1 10.09.09<br>
 +
-pMAlongLiFokA clone1 10.09.09<br>
 +
-pMAlongLiFoki clone1 10.09.09<br><br>
 +
-pMAFokA clone2 05.08.09 (old)<br>
 +
-pMAFoki clone2 05.08.09 (old)<br>
 +
-pMASplitFoki clone2 06.08.09 (old)<br>
-
<br>* Transformation with chemical and electro competent cells(RV 308):<br>
+
<br>*inoculation and plasmid preperation:
-
- did not work with chemical competent cells<br>
+
-pEX_strepFluA
-
- but worked with electro competent cells, absorption wavelength 400 nm (like CFP)<br>
+
<br>*Quenching test with HisFluASplitFoki and fluorescin-tagged oligos
-
(pictures coming soon, not finished jet)
+
<br>*BB Ago Pcr via Taq
 +
<br>*Started makin new VCS M13 Phages see Protocol day 1
-
<br>*Gel extraction with GelEx Kit from Quiagen: pEX-StrepDig, pEX-HisFluA, pEX-HisDig, short-linker Foki, middle-linker Foki, long-linker Foki
+
<h3>Plan for 04.10.09</h3>
-
<br>*Ligation with QuickLigase:
+
<br>*inoculation of
-
-short-linker Foki +pEx-HisDig/+pEX-HisFluA/+pEX-StrepDig
+
- pMA-Strep-Dig-LongLinker-Fok(inactive)<br>
-
-middle-linker Foki +pEx-HisDig/+pEX-HisFluA/+pExStrepDig
+
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)<br>
-
-long-linker Foki +pEx-HisDig/+pEX-HisFluA/+pEX-StrepDig
+
- pMA-Strep-Dig-ShortLinker-Fok(inactive)<br>
-
<br>*Transformation of Ligations with 100µl RV competent cells, 37°C room over night
+
- pMA-Strep-Dig-SplitLinker-Fok(inactive)<br>
-
<br>*sorting of right and wrong sequenced parts and parts which need subsequent cloning into a different vector
+
- pMA-His-Dig-SplitLinker_Fok(inactive)<br>
-
<br>*lab talk (Kristian, Tobias, Sven, Gerrit, Laura)
+
- pMA-His-Dig-SplitLinker_Fok(inactive)<br>
-
- DsbA-signalsequence for the exprot into the periplasma will be cloned to the Foka completed parts <br>
+
- pMA-Strep-Dig-SplitLinker-Fok(active)<br>
-
- in vivo experiment of e.colis cotransformated with Foka and Foki completed plasmids, elektroporated, transformated with M13 DNA ss with hybridized oligos, fluroescent oligos and without oligos<br>
+
- pMA-His-Dig-LongLinker-Fok(inactive)<br>
-
-> plaque essay will show efficency of Fok<br>
+
- pMA-His-FluA-LongLinker-Fok(inactive)<br>
-
- in vitro essay will be done with Fos fusioned to Foka, JUN fusioned to GFP and Foki added to experiment<br>
+
- pMA-His-FluA-MiddleLinker-Fok(inactive)<br>
 +
- pMA-His-FluA-ShortLinker-Fok(inactive)<br>
 +
- pMA-His-FluA-SplitLinker-Fok(inactive)<br>
 +
into LB-Amp
 +
<br>*digestion of pEx-Strep-Flua with AgeI and PstI
 +
<br>*digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive)
 +
<br>*ligation of the above parts and transformation into RV308
 +
<br>*plasmid preparation of...
-
<h3>22.09.09 Gerrit, Isabel, Sarah, Max, Hannes, Caro, Timo, Julia, Laura </h3>
+
-pMASplitFokA <br>
-
'''<br>*continued phage display:'''
+
-pMAFokA <br>
-
<br>*plasmid prep of pEX His Dig Rest Klone 6 pellet
+
-pMAFoki <br>
-
<br>*digest of pEX His Dig plasmid prep of 22.09 with ageI and pstI
+
-pMASplitFoki <br>
-
<br>*digest of pEX Strep Dig plsamidprep of 27.08 with ageI and pstI
+
-pMAshortLi<br>
-
<br>*digest of pEXHis FluA Split Foki 27.08 with xbaI and pstI , for rechieving a pEX vector
+
-pMAmiddelLi<br>
-
<br>*gel extraction of the digests
+
-pMAlongLi <br>
-
<br>*Quick ligation of pEXvector+HisFluA(gel Ex of 6.08.09) ; pEXHisDig+middle linker Foka(gel EX 11.09),
+
-pMAlongLiFokA <br>
-
pEXStrepDig+middle linker Foka(gel EX 11.09) ,pEXStrepDig+short linker Foka(gel EX 11.09)
+
-pMAlongLiFoki <br>
-
<br>*Transformation of the different constructs into RV308 bacteria
+
-pMAFokA (old)<br>
-
<br>*finishing of list of sequenced parts -> in folder SOPS
+
-pMAFoki (old)<br>
 +
-pMASplitFoki (old)<br>
-
<h3>23.09.09 Gerrit, Julia, Laura, Anika, Isabel </h3>
+
<h3>04.10.09, Laura, Anika, Max</h3>
 +
<br>* Plasmid preparation of
 +
-pMASplitFokA <br>
 +
-pMAFokA <br>
 +
-pMAFoki <br>
 +
-pMASplitFoki <br>
 +
-pMAshortLiFokA<br>
 +
-pMAshortLiFoki<br>
 +
-pMAmiddelLiFokA<br>
 +
-pMAmiddelLiFoki<br>
 +
-pMAlongLiFokA <br>
 +
-pMAlongLiFoki <br>
 +
-pMAFokA (old)<br>
 +
-pMAFoki (old)<br>
 +
-pMASplitFoki (old)<br>
 +
clone 1 and 2,respectively - results see notebook<br>
 +
<br>* Glycerolstock of <br>
 +
-pMASplitFokA <br>
 +
-pMAFokA <br>
 +
-pMAFoki <br>
 +
-pMASplitFoki <br>
 +
-pMAshortLiFokA<br>
 +
-pMAshortLiFoki<br>
 +
-pMAmiddelLiFokA<br>
 +
-pMAmiddelLiFoki<br>
 +
-pMAlongLiFokA <br>
 +
-pMAlongLiFoki <br>
 +
-pMAFokA (old)<br>
 +
-pMAFoki (old)<br>
 +
-pMASplitFoki (old)<br>
 +
clone 3
 +
<br>* Pellets of<br>
 +
-pMASplitFokA <br>
 +
-pMAFokA <br>
 +
-pMAFoki <br>
 +
-pMASplitFoki <br>
 +
-pMAshortLiFokA<br>
 +
-pMAshortLiFoki<br>
 +
-pMAmiddelLiFokA<br>
 +
-pMAmiddelLiFoki<br>
 +
-pMAlongLiFokA <br>
 +
-pMAlongLiFoki <br>
 +
-pMAFokA (old)<br>
 +
-pMAFoki (old)<br>
 +
-pMASplitFoki (old)<br>
 +
clone 1 and 2,respectively - stored in Pelletbox, -20°C
 +
<br>*Digest of
 +
1. pEXHisDigMiddleLFoki
 +
2. pMaScFaantiNIP
 +
3. prep of Ligation pEX+CAT of 09.07.09 old
 +
4. pMAShortLFoka<br>
 +
5. pMAShortLFoki<br>
 +
6. pMAMiddleLFoka<br>
 +
7. pMAMiddleLFoki<br>
 +
8. pMALongLFoka<br>
 +
9. pMALongLFoki<br>
 +
10.pEXStrepFlua<br>
 +
<br>*Preparative Gel of Digest<br>
 +
[[Image:Freiburg09_041009_umklonierung_gel.JPG|none|thumb|Agarose gel; Lanes: 1. pEXHisDigMiddleLFoki, 2. pMaScFaantiNIP, 3. pEX+CAT, 4. pMAShortLFoka, 5. pMAShortLFoki, 6. pMAMiddleLFoka, 7. pMAMiddleLFoki, 8. pMALongLFoka, 9. pMALongLFoki,10.pEXStrepFlua|400x400px]]
 +
Freiburg09_041009_umklonierung_gel.JPG
 +
<br>*Gelextraction<br>
 +
<br>*Ligation of<br>
 +
1.pMAHisDigMiddleFoki<br>
 +
2.pMACAT<br>
 +
3.pEXStrepFluAShortLFokA<br>
 +
4.pEXStrepFluAShortLFoki<br>
 +
5.pEXStrepFluAmiddleLFokA<br>
 +
6.pEXStrepFluAMiddleLFoki<br>
 +
7.pEXStrepFluALongLFokA<br>
 +
8.pEXStrepFluALongLFoki<br>
 +
9.JS 419StrepDigSplitFokA<br>
 +
10.JS 419HisDigSplitFokA<br>
 +
11.JS 418HisDigSplitFokA<br>
 +
Approach: 8µl H2O, 3 µl Vector, 6µl Insert, 2µl Quick Ligase Buffer, 1 µl Quick Ligase, 15 min, Room Temp.<br>
 +
<br>*Dephosphorylation of pBAD Vector: <br>
 +
Approach: 2µl Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 min. 37°C, 5 min. 75°C<br>
 +
<br>*Transformation of<br>
 +
1.pMAHisDigMiddleFoki (RV)<br>
 +
2.pMACAT(RV)<br>
 +
3.pEXStrepFluAShortLFokA(RV)<br>
 +
4.pEXStrepFluAShortLFoki(RV)<br>
 +
5.pEXStrepFluAmiddleLFokA(RV)<br>
 +
6.pEXStrepFluAMiddleLFoki(RV)<br>
 +
7.pEXStrepFluALongLFokA(RV)<br>
 +
8.pEXStrepFluALongLFoki(RV)<br>
 +
9.pBAD (RV)<br>
 +
10.pJS 419StrepDigSplitFokA(XBL)<br>
 +
11.pJS 419HisDigSplitFokA(XBL)<br>
 +
12.pJS 418HisDigSplitFokA(XBL)<br>
 +
<br>*test digest of plasmid preps from today
 +
-> run on a gel with digest pET39, xbaI, ecoRI from Tobi
 +
<br>*starter culture of pEx_DsbA_HisDigSplitFoka
 +
-> modified expression has to be done tomorrow
 +
<br>*inoculation of pJS418 and pJS419 for glycerolstocks tomorrow
-
'''Periplasma-project'''
+
<br>*Taq AGO BB PCR did not work
-
<br>*Transformation of plasmid vectors in XL1blue competent cells: Tt-; Aa in pet 28a; pet 39b
+
<br>*Phageproduction day 2
-
<br>*poured plates with Kan and Tet, 14 plates left in 4°c room
+
-
<br>*put in 37°c room over night
+
-
<br>*Digest of pMAStrep (prep of 09.07.09), pMAStrepFluA (prep of 06.08.09), pMAStrepDig (prep of 24.08.09) and pExStrepDig (prep of 27.08.09) with NgoMIV<br>
+
-
purpose: to get rid of snd NgoMIV restriction site
+
-
Plasmid: 10 µl<br>
+
-
water: 16.5 µl<br>
+
-
buffer: 3 µl buffer 4 NEB iGEM stock<br>
+
-
Restriction_enzyme_1 : 1 µl NgoMIVI from NEB KuKlabstock<br>
+
-
<br>*preparative gel and gel extraction
+
-
--> see picture
+
-
<br>*ligation and transformation<br>
+
-
<br>*plasmid prep of
+
-
...
+
-
<br>* phage display
+
-
- pelleted newly grown vector constructs (AGO)
+
-
<h3>24.09.09 Gerrit, Julia, Laura, Hannes, Caro </h3>
+
<h3>05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3>
-
<br>*plasmid preparation of
+
-
- pExHisDigMiddleliFoka<br>
+
-
- pExStrepDigMiddleliFoka<br>
+
-
- pExStrepDigShortliFoka<br>
+
-
- pExHisFluA<br>
+
-
-> glycerolstocks in new glycerolstocks in -80°C, pellets in -20°C in pellet box
+
-
<br>*SDS-gel of eluate, flow through and wash fractions of expressed and purified HisDigSplitFoka from 17.09.09
+
-
<br>*Western Blot of HisDigSplitFoka SDS Gel, incubated with anti His-Tag antibodies
+
-
--> no ECL signal
+
-
<br>*repetition of M13 ssDNA digest with oligos "Fokkontolle 2+3" and FokI --> didn't work
+
-
<br>*digest of pExHisFluASplitFoki (clon 2, 26.08.09) and pExStrepDigSplitFoka (clon 1, 29.08.09)
+
-
purpose: to ligate these parts in order to get a construct for the in vivo assay
+
-
Plasmid: 10 µl pExHisFluASplitFoki<br>
+
<br>* 3l LB and 2L LB Agar
-
water: 14.5 µl<br>
+
-
BSA: 0.5 µl NEB iGEM stock<br>
+
-
buffer: 3 µl buffer 2 NEB iGEM stock<br>
+
-
Restriction_enzyme_1 : 2 µl PstI from NEB KuKlabstock<br>
+
-
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
+
-
Plasmid: 10 µl pExStrepDigSplitFoka<br>
+
<br>*Periplasma Project
-
water: 14.5 µl<br>
+
-
BSA: 0.5 µl NEB iGEM stock<br>
+
-
buffer: 3 µl buffer 3 NEB iGEM stock<br>
+
-
Restriction_enzyme_1 : 1 µl PstI from NEB KuKlabstock<br><br>
+
-
<br>* inoculation<br>
+
-
- pEX Strep+Dig+Split+FokA 10ml LB+Amp<br>
+
-
- pEX_His+Dig+Split+FokA 10ml LB+Amp<br>
+
-
- pEX Strep+Dig 5ml LB+Amp<br>
+
-
- pMA_Strep+Dig 5ml LB+Amp<br>
+
-
- pMA_Strep 5ml LB+Amp<br>
+
-
- pMA_Strep+FluA 5ml LB+Amp<br><br>
+
-
<br>* Periplasma Project
+
<br>*digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09)
 +
<br>*test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka
 +
<br>*1) Gelextraction<br>
-
<br>*1) Inoculation
+
--> Gelbild <br>
-
- Pet39bXLblue in 100ml LB + 100µl KAN + 100µl TET<br>
+
1) Vector: pEXHisFluSplitFoki<br>
-
- aa in pet28aXL1blue in 100µl KAN + 100µl TET<br>
+
2)Insert: RBSStrepDigSplitFokA<br>
-
- Tt- XL1blue in 100µl KAN + 100µl TET<br>
+
3)PET39b+<br>
-
- Incubation (37°C) for 6 hours<br>
+
-
<br>*2)Transformation
+
<br>*2)Ligation<br>
-
--> XL1 blue pBadRDF<br>
+
-10ul 2 fold buffer<br>
 +
- 6 µl Insert<br>
 +
-3µl Vector<br>
 +
-1µl Quick Ligase<br>
-
-Plasmid DNA 0,6µl
+
--> 15 min at RT<br>
-
-cells 40 µl
+
-
-100 µl and rest platet out on AMP/Tet Plats
+
-
<br>* 3)Miniprep
+
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA<br>
-
- pJS419
+
-
- pJS418
+
-
- Concentrations determined with Nanodrop <br>
+
-
Nanodrop Data
+
<br>*3)Transformation<br>
 +
-5 µl DNA of Ligation + BL21d3 and XBL
-
[[Image:Freiburg09 24.09.09.jpg|none|thumb||400x400px]]
+
<br>*'''Mini prep with with Qiagen Spin Miniprep Kit of'''
 +
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br>
 +
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br>
 +
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Dbsa 1,2,3
 +
<br>*'''Made Glycerolstocks of'''
-
<br>*3) Testdigest
+
<br>*700µl Cellsuspension+ 300µl Glycerol
 +
- pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3<br>
 +
- pMA-His-Dig-SplitLinker_Fok(active)1,2,3<br>
 +
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3<br>
 +
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3<br>
 +
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3<br>
 +
- pMA-Dbsa 1,2,3
-
[[Image:Freiburg09 Periplamaproject Testverdau Sven 24-9001.jpg|none|thumb|1:pEX His Dig split FokA<br>
+
<br>*'''Test Digestion of'''
-
2:pEX Strep Dig split FokA<br>
+
-
-digested with: XbaI and NgoniV<br>
+
-
n: Fermentas GeneRuler DNA Ladder Mix |400x400px]]
+
-
-To 1) DNA  22,97 [µl]<br>
+
<br>*put in each sample: 5µl DNA, 2,5µl H2O, 1µl Buffer 3, 1µl EcoRI, 1µl PstI, 1µl BSA
-
NEB4 3,0[µl]<br>
+
-
XbaI 1,5[µl]<br>
+
-
NganIV 1,5[µl]<br>
+
-
BSA 0,5[µl]<br>
+
-
H20 0,53[µl]<br>
+
-
- To 2) DNA  21,97 [µl]<br>
+
- pMA-Strep-Dig-LongLinker-Fok(inactive)3<br>
-
NEB4 3,0[µl]<br>
+
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1<br>
-
XbaI 1,5[µl]<br>
+
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1<br>
-
NganIV 1,5[µl]<br>
+
- pMA-Strep-Dig-SplitLinker-Fok(inactive)3<br>
-
BSA 0,5[µl]<br>
+
- pMA-His-Dig-SplitLinker_Fok(inactive)1<br>
-
H20 2,25[µl]<br>
+
- pMA-His-Dig-SplitLinker_Fok(active)2<br>
 +
- pMA-Strep-Dig-SplitLinker-Fok(active)1<br>
 +
- pMA-His-Dig-LongLinker-Fok(inactive)1<br>
 +
- pMA-His-FluA-LongLinker-Fok(inactive)3<br>
 +
- pMA-His-FluA-MiddleLinker-Fok(inactive)3<br>
 +
- pMA-His-FluA-ShortLinker-Fok(inactive)1<br>
 +
- pMA-His-FluA-SplitLinker-Fok(inactive)2<br>
 +
- pMA-Dbsa 2<br>
-
- Purification: 0,8% Agarosegel<br>
+
<br>* Transformation of<br>
-
- Quigen Gelex SpinKit<br>
+
- XBL pEXHisFluASplitFokIRBSStrepDigSplitFokA<br>
 +
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA<br>
 +
<br>*Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each
 +
- induced with 0,7mM IPTG and took samples T0-T5<br>
 +
- centrifuged in buckets 17', 4000rpm, 4°C<br>  
 +
- eluted in 20 ml TES in each bucket<br>
 +
....
-
<h3>25.09.09 Gerrit,Laura,Hannes,Isabel,Max,Anika,Caro, Timo </h3>
+
<br>*PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones
 +
<br>*PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature
-
<br>*Periplasma Project
+
<h3>06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika</h3>
 +
<br>*digest
 +
- pBAD vector <br>
 +
DNA: 16.8µl<br>
 +
enzyme: 1µl AgeI<br>
 +
buffer: 2µl buffer 1 <br>
-
1) Ligation with Quick Ligase from NEB<br>
+
- pBAD insert <br>
 +
DNA: 16.8µl<br>
 +
enzyme: 1µl XmaI<br>
 +
buffer: 2µl buffer 4 <br>
 +
BSA (10X): 2µl <br>
-
- 2fold Quick Ligase Buffer 10µl<br>
+
- pJS 418/419 <br>
-
- DNA ( Insert(0,5µl)+Vector(9µl))<br>
+
DNA: 10µl<br>
-
- Ligase 1µl<br>
+
enzyme: 1µl PstI and 1.5 µl Xba/<br>
 +
buffer: 3µl buffer 3 <br>
 +
BSA (10X): 3µl <br>
-
-10 min Ligation at RT<br><br>
+
- pExStrepDigSplitFoka/pExHisDigSplitFoka <br>
 +
DNA: 16.8µl<br>
 +
enzyme: 1µl PstI and 1.5 µl Xba/<br>
 +
buffer: 3µl buffer 3 <br>
 +
BSA (10X): 3µl <br>
-
2) Miniprep <br>
+
<br>*testdigest of pExHisFluASplitFoki_StrepDigSplitFoka
 +
DNA: 5µl<br>
 +
enzyme: 1µl PstI and 1.5 µl Xba/<br>
 +
buffer: 1µl buffer 3 <br>
 +
BSA (10X): 1µl <br>
 +
<br>*preparative gels of digests
 +
<table border="0">
 +
<tr><td>
 +
[[Image:Freiguburg09_061009_bad_js.JPG|none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418|300x300px]]
 +
</td>
 +
<td>
 +
[[Image:Freiburg09_061009_js_foka.JPG |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
 +
</td></tr></table>
 +
interpretation: pExStrepDigSplitFoka seemed to be a wrong construct, the other constructs showed bands of the right size even if the concentration of the PCR pBAD insert seems to be very low
 +
<br>*testdigest of pMAdsba
 +
DNA: 5µl<br>
 +
enzyme: 1µl PstI and 1.5 µl Xba/<br>
 +
buffer: 1µl buffer 3 <br>
 +
BSA (10X): 1µl <br>
-
pEX His Dig Split FokA<br>
+
<br>*His-tag purification of HisDigSplitFoka (periplasm export) with Ni-NTA column. Washing buffer: 25mM imidazole
 +
-cells were sonicated for 2 x 1min before filtering with 0.45µm and 0.22µm filter
-
pEX Strep Dig Split FokA<br>
+
<br>*Ligation
 +
- Dephphorylation of pBAD Gelex<br>
 +
- 1µl Fast AP<br>
 +
- 5.5µl fast AP buffer<br>
 +
-1.5µl water<br>
-
Nanodrop Data
+
--> Solution was given to eluat
-
[[Image:Freiburg_09 Nanodrop 25-9.jpg |none|thumb|The two last lines only|400x400px]]<br><br>
+
-
Stored: Plasmidbox in freezer<br>
+
- for each ligation:<br>
-
<br>* Amp preparation, -20°C<br>
+
- 6µl Insert<br>
-
<br>* Plasmidprep<br>
+
-3µl Vetor<br>
-
- pEXStrep+Dig Clone 1+2  (Tube 1+2)<br>
+
-1µl Quickligase<br>
-
- pMA_Strep+Dig Clone 1+2  (Tube 3+4)<br>
+
-10µl buffer<br>
-
- pMA_Strep Clone 1+2 (Tube 5+6)<br>
+
-
- pMA_Step+FluA Clone 1+2 (Tube 7+8)<br>
+
-
--> -80°C<br><br>
+
-
<br>* pellets of:<br>
+
-
- pEX_Strep+Dig Clone 3+4  (Tube 9+10)<br>
+
-
- pMA_Strep+Dig Clone 3+4  (Tube 11+12)<br>
+
-
- pMA_Strep Clone 3+4 (Tube 13+14)<br>
+
-
- pMA_Step+FluA Clone 3+4 (Tube 15+16)<br>
+
-
--> -20°C<br><br>
+
-
<br>* Transformation of<br>
+
--> pBAD <br>* Insert (Dummy)<br>
-
pEX_His+FluA+Split+Foki+Strep+Dig+Dig+Split+FokA into RV308 and XBL<br>
+
--> pJS419+HisDigSplitFoka
 +
-->pJ418+HisDigSplitFoka
 +
<br>* Transformation
-
<br>* phage display
+
--> pBAD <br>* Insert (Dummy)<br>
-
- error prone PCR<br>
+
--> pJS419+HisDigSplitFoka
-
water: 29,3µl<br>
+
-->pJ418+HisDigSplitFoka
-
DNA: 1,8µl<br>
+
-
buffer: 5µl (10x, without MgCl2)<br>
+
-
primers (87/88 or 87/89): 1,5µl each<br>
+
-
dNTPs: 1,5µl (10mM each)<br>
+
-
Taq: 1µl<br>
+
-
MgCl2: 12µl (25mM)<br>
+
-
MnCl2: 5µl (5mM)<br>
+
-
<h3>26.09.09 Caro </h3>
+
- in XLblue<br>
-
<br>* Inoculation
+
-
- 4 clones respectively of:<br>
+
-pBAD was plated on AMP plates<br>
-
-pEX His dig ML FokA <br>
+
- Both pJS... were plated on CM plates<br>
-
-pEX Strep Dig SL FokA <br>
+
-
-pEX His FluA<br>
+
-
-pEX Strep Dig ML FokA <br><br>
+
-
-pEX His FluA Split Foki Strep Dig Split FokA aus RV308<br>
+
<br>*'''Ligation and Transformation of'''
-
-pEX His FluA Split Foki Strep Dig Split FokA aus XL<br>
+
-
- Medium: <br>
 
-
- 200 ml LB <br>
 
-
- 2g Glucose (sterile filtered)<br>
 
-
- 200 µl AMP<br><br>
 
-
<br>* new DsbA SS insertion in front of FokA constructs<br>
+
Ligation:per sample 8µl H2O; 3µl vector; 6µl insert; 2µl Quick Ligase buffer; 1µl Quick Ligase
-
- 3 h digestion of pEX_His_Dig_Split_FokA (prep 25.9.) & pEX_Strep_Dig_Split_FokA using 1.5 µL XbaI, NgoMIV in 30 µL final<br>
+
-
- 0.8 % agarose gel<br>
+
-
- GelEx, 30 µL elution<br>
+
-
- Quickligation using 1:3 ratio, 10'<br>
+
-
- transformed XL1 & BL21 chemically using 10 µL ligation rx. (low conc.) & 50 µL cells<br>
+
-
<br>
+
-
<h3>27.09.09 Anika, Hannes </h3>
 
-
<br>* Miniprep
 
-
[[Image:Freiburg09_270909_Minipreps.jpg|none|thumb||600x600px]]
 
 +
Transformation:<b>Defrost competent cells on ice:</b>(100 µl);
 +
<b>add of the ligation:</b> 5 µl;
 +
<b>DNA and cells:</b> mix softly by knocking;
 +
<b>Incubation on ice for:</b> 20-30 min;
 +
<b>Heat shock at:</b> 42°C for 40 sec;
 +
<b>Cool off on ice for:</b> 5 min;
 +
<b>add sterile LB(or dyt)medium:</b> 900 µl;
 +
<b>Incubation in(shaker)at:</b> 37°C for 60-70 min;
 +
<b>Plate cells on LB+antibiotic plates:</b> ampicillin;
 +
<b>ligation:</b> 2 plates: 1. 50µl cells
 +
<b>2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out:</b>
 +
-pMA HisDig Middle Linker Foki<br>
 +
-pMA CAT<br>
 +
-pEX Strep FluA SL FokA<br>
 +
-pEX Strep FluA LL Foki<br>
 +
-pEX Strep FluA ML Foki<br>
 +
-pEX Strep FluA ML Foka<br>
 +
-pEX Strep FluA LL Foka<br>
-
<br>* test digest of DsbA ss insertion
 
-
- 800 ng DNA, 0.5 µL AvaI & XbaI, 15 µL total, 30'<br>
 
-
- 2 % high-res agarose gel, 100 bp ladder (expected: 237 bp for His, 242 bp for Strep, failure = 184 bp)
 
-
<br>* all new DsbA insertion transformations successful, control plate empty
+
<br>*Sequencing:
-
--> o/n cultures in LB (Glu, Amp), 4 each (DsbA_His_Dig_Split_FokA & DsbA_Strep_Dig_Split_FokA, XL1 & BL21)<br>
+
-
<br>*Inoculation of starter culture for expression
+
27µl H2O; 3µl DNA
-
-pExHisFluASplitFoki in BL21de3 (300ml LB Amp, 1% glucose)<br>
+
-
--> 26°C
+
-
<br>*inoculation of
+
-pMA Dbsa clone 2<br>
-
-pEx_DsbAss_HisDigSplitFoka in XL1<br>
+
-pMA HisDigSplitFokA clone 2<br>
-
-pEx_DsbAss_StrepDigSplitFoka in XL1<br>
+
-pMA HisFluASL Foki clone 1<br>
-
-pEx_DsbAss_HisDigSplitFoka in BL21<br>
+
-pMA HisFluASplitFoki clone 2<br>
-
-pEx_DsbAss_StrepDigSplitFoka in BL21<br>
+
-pMA StrepDigLLFoki clone 3<br>
 +
-pMA HisFluaMLFoki clone 3<br>
 +
-pMA StrepDigSplitFokA clone 1<br>
 +
-pMA StrepDigMLFoki clone 1<br>
 +
-pMA StrepDigSLFoki clone 2<br>
 +
-pMA HisDigLLFoki clone 1<br>
 +
-pMA StrepDigSplitFoki clone 3<br>
 +
-pMA HisDigSplitFoki clone 1<br>
 +
-pMA HisFluaLLFoki clone 3<br>
-
in 5ml LB Amp, 1% glucose
+
<br>*Inoculation
-
<h3>28.09.09 Isabel, Sarah, Gerrit, Anika, Hannes, Timo, Caro, Max </h3>
+
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight<br>
-
<br>*plasmid preparation of pEX_DsbAss-His-Dig-Split-FokA (clone 1-5) and pEX-DsbAss-Strep-Dig-Split-FokA (clone 1-5)
+
<br>*Yesterdays improved pcr resulted in a lot of product, but still unspecific ones.. made new one with even higher annealing temperature (69°C) and a bit less cycles (35)
-
<br>*continued phage display
+
 
-
- plasmid preparation of pJs#440 <br>
+
<br>*New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 and A4 _see gele_
-
- error prone PCR<br>
+
no differend results were obtained compaired to the assays using unphorphorylated oligos
-
water: 21,75µl<br>
+
 
-
template: 1,5µl<br>
+
<br>*Started dialysis to transfer the  leftover AGO-proteins into the assay buffer
-
primers (87/88 or 87/89): 1,5µl (10µM) each<br>
+
 
-
dNTPs: 1,5µl (10mM each)<br>
+
<h3>07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika</h3>
-
Taq: 1µl<br>
+
<br>*SDS gel of protein purification of HisDigSplitFoka (periplasm) from 06.10.09
-
buffer(10x): 5µl<br>
+
 
-
MnCl2: 5µl (5mM)<br>
+
 
-
MgCl2: 12µl (25mM)<br>
+
[[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker broad range, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution fraction 6, flow through fraction 3, washing fraction 2, periplasm extract (frozen over night at -80°C)|400x400px]]
-
-> 4 samples of 87/88 and 87/89 each<br>
+
 
-
water: 26µl<br>
+
 
-
pcr-product ("template"): 2µl<br>
+
<br>* Inoculation
-
primers (87/88 or 87/89): 1,5µl each<br>
+
4 Clones respectively:<br>
-
dNTPs: 1,5µl (10mM each)<br>
+
- pMA-His-Dig-MiddleLinker-Foki in XLBlue<br>
-
Taq: 1µl<br>
+
- pJS419-HIs-Dig-Split-Foka-  in XLBlue<br>
-
buffer: 5µl<br>
+
- pJS418-HIs-DIg-Split-Foka-  in XLBlue<br>
-
MnCl2: 2,5µl<br>
+
- pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest<br>
-
MgCl2: 9µl<br>
+
- pEX-Strep-FluA-LongLinker-Foki in XL1blue rest<br>
-
-> 4 samples of 87/88 and 87/89 each<br>
+
- pMA-CAT in XLblue 10µl<br>
 +
-pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest<br>
 +
 
 +
2 Clones respectively:<br>
 +
-pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09
 +
- pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09
 +
 
 +
 
 +
<br>* Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3
 +
<br>*two step PCR assembly of DsbA, His_Fos, and SplitFoka
 +
- program name: Assembly<br>
 +
- three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step
 +
<br>*preparative gel of the PCR samples
 +
-> primer haven't been diluted an probably made all secondary structures, has to be repeated
 +
<br>*digest of pBAD with AgeI and new PCR with insert digested with XmaI
 +
<br>*preparative gel with digest and
 +
<br>*test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka
 +
DNA: 5µl<br>
 +
Enzymes: 0.5µl of BamHI and MfeI each<br>
 +
Buffer: 1µl buffer 4<br>
 +
BSA (10fold): 1µl<br>
 +
Water: 2µl<br>
 +
-> pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1<br>
 +
<br>*plasmidpreparation of
 +
- pEXHisFluASplitFoki<br>
 +
- pExStrepDigSplitFoka<br>
 +
-> low concentrations<br>
 +
<br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka
 +
pExHisFluASplitFoki: 15µl<br>
 +
Enzymes: 1µl of PstI and 1.5µl SpeI<br>
 +
Buffer: 3µl buffer 2<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 9µl<br>
 +
 
 +
pExStrepDigSplitFoka: 15µl<br>
 +
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 3<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 9µl<br>
 +
 
 +
pExStrepDigSplitFoka: 10µl<br>
 +
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 3<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 14µl<br>
 +
<br>* Gelextraction of digest
 +
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) <br>
 +
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br>
 +
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br>
 +
 
 +
 
 +
<br>*preparative gels of digests
 +
<table border="0">
 +
<tr><td>
 +
[[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]]
 +
</td>
 +
<td>
 +
[[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
 +
</td></tr></table>
 +
 
 +
RBS PCR Product: m= 90 mg<br>
 +
vector: 0 0 130 mg<br>
 +
 
 +
<br>* Ligation
 +
- 6µl Insert<br>
 +
-3µl Vetor<br>
 +
-1µl Quickligase<br>
 +
-10µl buffer<br>
 +
 
 +
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br>
 +
<br>*Transformation
 +
 
 +
- All in XL1blue<br>
 +
- Vector: pEX-strep-Duig-Split-Foka<br>
 +
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka<br>
 +
 
 +
<br>*Stardet production of Phages baering the phagmid vektors with pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml each. see phageproduction protocoll day 1
 +
<br>*prepaired ELISA with anti-flag antibodies
 +
<br>*made electro competent cells for transformation with the ago phagmidbibliothek
 +
 
 +
<h3>08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes </h3>
 +
<br>*Poured SDS-Gels<br>
 +
<br>*Phage ss DNA
 +
 
 +
-Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet<br>
 +
-Transfomation with 3µl M13 Phage stock (-80°) at 10:30Uhr , incubated for 4-5 hours at 37°C in shaker<br>
 +
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C<br>
 +
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4°C room<br>
 +
 
 +
<br>*Miniprep
 +
 
 +
- 2 clones respectively:<br>
 +
-A = pEX strep dig split foka
 +
 
 +
<br>*Finished Phage production(see protocol day 2): We obtained approximately 3.8<br>*10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1<br>*10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of them to the anti-Flag ELISA<br>
 +
 
 +
<br>*Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):<br>
 +
see 405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to positive control (G5-6) and negative control (G3-4 and D3-10) detected about half an hour after the ABTS substrat was addet.
 +
 
 +
<>    1     2    3    4    5    6    7    8    9    10    11    12    <br>
 +
A      0.0470 0.0420 0.0440 0.0430 0.0480 0.0510 0.0460 0.0470 0.0470 0.0480 0.0450 0.0460<br>
 +
B      0.0470 0.0460 0.0480 0.0480 0.0460 0.0450 0.0470 0.0560 0.0470 0.0450 0.0450 0.0440<br>
 +
C      0.0460 0.0470 0.0500 0.0450 0.0430 0.0460 0.0450 0.0460 0.0430 0.0420 0.0440 0.0470<br>
 +
D      0.0490 0.0470 0.1590 0.1610 0.1920 0.1720 0.2210 0.2500 0.2130 0.2680 0.0460 0.0480<br>
 +
E      0.0490 0.0490 0.0450 0.0490 0.0430 0.0490 0.0460 0.0430 0.0460 0.0420 0.0450 0.0440<br>
 +
F      0.0490 0.0430 0.0470 0.0440 0.0470 0.0470 0.0440 0.0450 0.0500 0.0460 0.0450 0.0450<br>
 +
G      0.0500 0.0440 0.2530 0.2510 3.9180 3.8800 0.9560 0.9380 1.2420 1.2360 0.0440 0.0460<br>
 +
H      0.0480 0.0430 0.0510 0.0470 0.0480 0.0460 0.0470 0.0450 0.0450 0.0440 0.0460 0.0460<br>
 +
 
 +
<br>* Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction
 +
 
 +
<br>*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka
 +
pExHisFluASplitFoki: 15µl<br>
 +
Enzymes: 1µl of PstI and 1.5µl SpeI<br>
 +
Buffer: 3µl buffer 2<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 9µl<br>
 +
 
 +
pExHisDigSplitFoka: 15µl<br>
 +
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 3<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 14µl<br>
 +
 
 +
RBSStrepDigSplitFoka: 5µl<br>
 +
Enzymes: 1µl of XbaI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 3<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 19µl<br>
 +
->made two digest of PCR construct<br>
 +
<br>* Gelextraction of digest
 +
1) Insert: His-Dig-Split-Foka( digested with xba and pst) <br>
 +
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)<br>  
 +
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)<br>
 +
and also PCR purification of RBS-Strep-Dig-Split-FokA
 +
<br>*preparative gels of digests
 +
->see picture<br>
 +
<br>*ligation of
 +
-pEX-His-Flua-Split-Foki_His-Dig-Split-Foka<br>
 +
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)<br>
 +
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)<br>
 +
<br>*transformation of ligations
 +
<br>*inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol
 +
<br>*new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel
 +
->interpretation: no band of 921bp was visible<br>
 +
<br>*one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel
 +
->see picture<br>
 +
->interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible<br>
 +
<br>*new one step PCR of pMADsba with newly prepared 1:1000 dilution
 +
<br>*test digest of plasmidpreparations from today
 +
->see picture<br>
 +
 
 +
<h3>09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika </h3>
 +
 
 +
<br>*Phage ss DNA<br>
 +
-Over night in 4°C room precipitated phages: centrifuged for 20 min at 5000rpm and 4°C<br>
 +
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet recognized)<br>
 +
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at 13000rpm<br>
 +
-Decant the supernatant into new eppis, precipitate with 170µl PEG/NaCl and leave for 1 hour on ice<br>
 +
 
 +
<br>*Miniprep
 +
- pJS 419-his-dig-split-foka (clon 1+2)<br>
 +
- Glycerolstock of clon1 and 2<br>
 +
- 300µl Glycerol<br>
 +
- 700 µl culture<br>
 +
 
 +
- Pellets from clones 3,5,6<br>
 +
 
 +
Nanodrop data:<br>
 +
pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl<br>
 +
pJS 419-his-dig-split-foka-clon2 =468 ng/µl<br>
 +
 
 +
<br>*Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
 +
<br>*send to sequencing:<br>
 +
pMA Dig Plasmidprep 22.07.09 Timo1<br>
 +
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2<br>
 +
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3<br>
 +
pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4<br>
 +
pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5<br>
 +
pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6<br>
 +
pMA Cat plasmidprep clone1 08.10.09 Timo7<br>
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8<br>
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9<br>
 +
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12<br>
 +
 
 +
<br>* concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation
 +
<br>* electrical trafo of the 449 ago phagmid bibliothek into XL1
 +
<br>* prepaired immutubes with streptavidin for ago phages test panning tomorrow
 +
 
 +
 
 +
 
 +
<h3>10.10.09 Julia, Caro, Laura, Christoph</h3>
 +
 
 +
<br>*Measured OD600 of over night ER2738 culture abs.1)0.218 (1:10);2)0.23 (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet<br>
 +
<br>*Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM
 +
<br>*Growed Er2738 up to OD600 abs.0.6  8x 250ml in 1L Erlenmeyer flasks and infected with 15µl M13 phage, shake for 4 hours at 37°C
 +
<br>*Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4°C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl
 +
<br>*Precipitaion over night in 4°C room
 +
<br>*plasmidpreparation of
 +
- pBADQuick clones 1-6<br>
 +
- pBADT4 clones 1-6<br>
 +
- pMAYFP clone 1-2<br>
 +
- pMACFP clone 1<br>
 +
- pMASplitlinker clones 1-2<br>
 +
- HisTag clones 1-2 (but test digest was negative -> thrown away)<br<
 +
<br>*digest for recloning of pMA constructs:
 +
 
 +
pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from 10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09, pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl<br>
 +
Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 1<br>
 +
BSA (10fold): 3µl<br>
 +
Water: 10,5µl<br>
 +
 
 +
pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from 25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from 01.10.09: 10µl each<br>
 +
Enzymes: 1µl of AgeI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 1<br>
 +
BSA (10fold): 3µl<br>
 +
Water: 10,5µl<br>
 +
<br>*PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together
 +
<br>*analytical gel of PCRs -> didn't work
 +
<br>*preparative gels of digests
 +
-> see picture
 +
-> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more
 +
<br>*ligation of
 +
-pMAStrepDigShortLiFoka<br>
 +
-pMAStrepDigMiddleLiFoka<br>
 +
-pMAStrepDigLongLiFoka<br>
 +
-pExStrepDigShortLiFoka<br>
 +
-pExStrepDigMiddleLiFoka<br>
 +
-pExStrepDigLongLiFoka<br>
 +
-pMAHisDigShortLiFoka<br>
 +
-pMAHisDigMiddleLiFoka<br>
 +
-pMAHisDigLongLiFoka<br>
 +
-pMACATNd4 (MQI)<br>
 +
-pMACATNd4 (MQII)<br>
 +
-pMA Kontrolle (M)<br>
 +
-> in XLBlue<br>
 +
- pEx_CATNd4 (EQI)<br>
 +
- pEx_CATNd4 (EQII)<br>
 +
- pEx_Kontrolle(E)<br>
 +
->RV308
 +
Insert: 6µl <br>
 +
vector: 3µl <br>
 +
Ouick ligase buffer: 10µl <br>
 +
Quick ligase: 1µl <br>
 +
 +
<br>*transformation of ligations in XBL on LB/Amp/1%glucose plates
 +
<br>*cotransformation of
 +
<br>*testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture<br>
 +
[[Image:Freiburg09 101009pet39b+ssdnaagoassay.JPG|none|thumb|gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA|400x400px]]
 +
<br>*cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates
 +
<br>*inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09
 +
 
 +
<h3> 11.10.09, Timo, Hannes, Max, Anika</h3>
 +
 
 +
<br>* Digestion of
 +
1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI<br>
 +
2.pMAFos_HlsbZip, XbaI - AgI<br>
 +
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI<br>
 +
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br>
 +
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI<br>
 +
6.pMA_BB057, XbaI - PstI (01.10.09)<br>
 +
<br>*gel extraction of
 +
- pMA<br>
 +
- pEx<br>
 +
- ...
 +
 
 +
<br>* Testdigestion of
 +
7.pMA_YFP 2 (10.10.09, Caro)<br>
 +
8.pMA_CFP (10.10.09, Caro)<br>
 +
9.pMASplitLi1 (10.10.09, Caro)<br>
 +
10.pMASplitLi2 (10.10.09, Caro)<br>
 +
...image..
 +
<br>* 1% Agarosegel of constructs above
 +
<br>* Phage breeding day 2
 +
<br>* Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3)
 +
 
 +
<br>* plasmid prep of pEx_DsbA_StrepDigSplitFoka
 +
<br>* plasmid prep of pEx_HisDigSplitFoka
 +
 
 +
<br>* M13 ssDNA produced with bacterial of Julia and tried another variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected with <br> M13phage particles and let it grow for 2h. After this followed the qiagen M13 protocol for M13Dna
 +
 
 +
<h3> 12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit</h3>
 +
<br>* protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22°C.
 +
<br>* made 5 litres of DYT
 +
<br>*digest of pMASplitFoka clone 1 and 2 from 04.10.09
 +
DNA:10µl
 +
Enzymes: 1µl of NgoMIVI and 1.5µl PstI<br>
 +
Buffer: 3µl buffer 1<br>
 +
BSA (100fold): 0.5µl<br>
 +
Water: 14.5µl<br>
 +
<br>* Plasmidprep. of: <br>
 +
1.pMAFokA (old)<br>
 +
2.pMA-CAT Ndelta4 (MQII)1<br>
 +
3.pMA-CAT Ndelta4 (MQII)2<br>
 +
4.pMA-CAT Ndelta4 (MQII)3<br>
 +
5.pMA-CAT Ndelta4 (MQII)4<br>
 +
6.pMA-CAT Ndelta4 (MQII)5<br>
 +
7.pMA-CAT Ndelta4 (MQII)6<br>
 +
8.pMAFoka clone 1 from 04.10.<br>
 +
<br>*Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen...
 +
<br>*Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay
 +
<br>*Made digest of errorprone PCR product of the AGO-G3P constructs (from 02.10.; one with, one without Amber) via NheI and SfiI to gain new Phagmid library
 +
 
 +
<br>*Send to sequencing:[[Protocols#DNA_Sequencing]]<br>
 +
Julia 1-6:<br>
 +
1)pBADQuick clone 3<br>
 +
2)pBAD T4 clone 2<br>
 +
3)pBAD T4 clone 1<br>
 +
4)pMA YFP clone 1<br>
 +
5)pMA CFP clone 1<br>
 +
6)pMA Split Linker clone 2<br><br>
 +
 
 +
Gerrit1: pMA_cat-Nd4
 +
 
 +
<br>*Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for Panning Simulation for tomorrow
 +
<br>*made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki
 +
->will prepare electrocompetent cells tomorrow<br>
 +
<br>*inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp
 +
<br>*PCR of pMADsba repeated with different (higher) template dilutions
 +
->analytical gel (see picture)<br>
 +
->didn't work again<br>
 +
<br>*poured IPTG/XGal plates for in vivo plaque assay test run
 +
 
 +
<br>*transformation of:<br>
 +
- pBAD_FokA (Quick)<br>
 +
- pBAD_CAT(Quick)<br>
 +
- pBAD_FokA (T4)<br>
 +
- pBAD_CAT (T4)<br>
 +
- pEX_FokA+YFP<br>
 +
- pEX_DsbA+FokA+YFP<br>
 +
into BL21 de3 gold<br>
 +
Plates: LB+ AMP +1%Glucose<br>
 +
<br>*cultivation of
 +
pma strep dig split foka<br>
 +
pma long linker<br>
 +
pma his flua split foki<br>
 +
 
 +
 
 +
<h3> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia</h3>
 +
 
 +
<br>*Miniprep
 +
 
 +
-pMA-fos 1<br>
 +
-pMA-fos 2<br>
 +
-pMA his flua split fokI<br>
 +
- pMA strep dig split foka <br>
 +
-pMA long linker<br>
 +
 
 +
<br>*testdigest of Plasmidprep from today
 +
 
 +
15 µl for 6µl loading dye
 +
-buffer 2 1µl<br>
 +
-xbaI 0.5µl<br>
 +
-pstI 0.75µl<br>
 +
-DNA 2 µl<br>
 +
-water 9.75µl<br>
 +
-BSA 1µl<br>
 +
 
 +
--> Gelbild
-
<br>*transformation of pEx_HisFluASplitFoki in BL21de3
+
- Glycerolstocks are in in box of 5/10/2009
 +
<br>*made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka)
 +
->stored in -80°C
 +
<br>*culture of ER2738 in LB Tet for cotransformation in afternoon
 +
<br>*test in vivo assay
 +
- electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV<br>
 +
- 1.5h on 37°C shaker at 750rpm<br>
 +
- precipitation of phages<br>
 +
- infection of ER2738 with different dilutions of phages<br>
 +
- mixed cells with Top agar and plated them on IPTG/XGAL plates -> 37° shaker<br>
<br>*inoculation of
<br>*inoculation of
-
-pEx_HisDigSplitFoka in XL1blue (?)<br>
+
-pMASplitFoka clone 1 from prep 04.10.09<br>
-
-pEx_StrepDigSplitFoka in XL1blue (?)<br>
+
-pMAStrepDig clone 2 from prep 25.09.09<br>
 +
-pMAMiddleFoka clone 2 from prep 04.10.09<br>
 +
-pMAShortFoka clone 2 from prep 04.10.09<br>
 +
-pMAFoka clone 1 from prep 04.10.09<br>
 +
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09<br>
 +
-pMALongFoka clone1 prep from 10.09.09<br>
 +
-pMAFos 13.10.09<br>
 +
-pBAD_CAT 13.10.09<br>
 +
-pBAD_Foka 13.10.09<br>
 +
-pMADsbAHisDigSplitFoka 13.10.09<br>
 +
-pMADsbAStrepDigSplitFoka 13.10.09<br>
 +
-pExFosSplitFoka 13.10.09<br>
 +
-pMAFosSplitFoka 13.10.09<br>
 +
<br>*protein purification of DsbA_StrepDigSplitFoka with Strep column
-
<h3>29.09.09 Gerrit, Anika, Hannes, Caro, Laura, Christoph, Max </h3>
+
<br>*digestion, 1% agarose gel and gel extraction of
-
<br>*Identified the mutation that was revealed by the latest sequencing: All plasmids bared a mutation that led to an exchange in amino acid sequence: 371 L->S. The aa 371 (indicated in pink on the right) is part of an peripheral alpha helix in the mid-domain of the argonaute protein and has no contacts to aa outside of the alpha helix and should the thereby not have major influence of the structure or function of the protein.
+
- pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br>
-
[[Image:Freiburg 09 Mutation in AGO.png|none|thumb||600x600px]]
+
- pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI<br>
-
<br>*because the ligation pExHisFluASplitFoki_StrepDigSplitFoka didn't work a new digest of pExHisFluASplitFoki and pExStrepDigSplitFoka was done:
+
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI => wasn't ok on the gel, inoculated clone 1<br>
-
Plasmid: 10 µl pExHisFluASplitFoki, clone 1 (100µl) from 26.08.09<br>
+
- pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br>
-
water: 14.5 µl<br>
+
- pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br>
-
BSA: 0.5 µl NEB iGEM stock<br>
+
- pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br>
-
buffer: 3 µl buffer 2 NEB iGEM stock<br>
+
- pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br>
-
Restriction_enzyme_1 : 2 µl PstI from NEB KuKlabstock<br>
+
- pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br>
-
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock <br>
+
- pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br>
 +
- pEx-Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI
 +
- pMA (01.10., c=500ng/µl) with XbaI + AgeI
 +
- pMA (01.10., c=500ng/µl) with EcoRI + SpeI
-
Plasmid: 10 µl pExStrepDigSplitFoka, clone from 29.08.09<br>
 
-
water: 14.5 µl<br>
 
-
BSA: 0.5 µl NEB iGEM stock<br>
 
-
buffer: 3 µl buffer 3 NEB iGEM stock<br>
 
-
Restriction_enzyme_1 : 2 µl XbaI from NEB KuKlabstock<br>
 
-
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
 
-
->put in 37°C for 2h
 
-
<br>*dephosphorylation of vector pExHisFluASplitFoki by SAP
 
-
DNA: 30µl<br>
 
-
SAP: 0,5µl<br>
 
-
SAP buffer: 3,5µl<br>
 
-
water: 1µl<br>
 
-
- 37°C for 30min<br>
 
-
- 70°C for 20min<br>
 
-
- PCR purification Kit (normally direct digest would have been sufficient<br>
 
-
<br>*preparative gel of digest to gain vector and insert
 
-
[[Image:Freiburg09_290909_foki_foka_verdau.JPG|none|thumb|Agarose gel; Lanes: 1.pExHisFluASplitFoki, 2.pExStrepDigSplitFoka ||  400x400px]]
 
-
<br>*gel extraction
+
<br>*ligations:
-
<br>*ligation of pExHisFluASplitFoki and StrepDigSplitFoka, pExHisFluASplitFoki (as negativ control)
+
Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI<br>
-
<br>*transformation of pExHisFluASplitFoki_StrepDigSplitFoka and pExHisFluASplitFoki (negative control)
+
Insert: 6 µl LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI<br>
-
<br>*PCR primer forward and reverse for the in vivo assay/ polycistronic assay arrived
+
Buffer: 10 µl, QuickLigase Buffer NEB<br>
-
-> PCR with pEXStrepDigSplitFoka was done to amplificate the gene and insert a RBS site simultanously
+
QuickLigase: 1 µl<br><br>
-
<br>*Inoculation of starter culture for expression and glycerol stocks:
+
-
- pEx_HisFluASplitFoki in BL21de3 (300ml 2YT Amp, 1% Gluc) --> 26°C<br>
+
-
<br>*Inoculation of
+
-
- pET-39b+ in XL1blue (15ml LB+Kan) --> plasmidprep for cleavage assays with AGO tomorrow<br>
+
-
--> 37°C over night<br>
+
-
<br>*sequencing of HisFluASplitFoki clone 1 (100µl) from 26.08.09
+
-
<h3>30.09.09 Gerrit, Hannes, Caro, Anika, Max, Isabel, Christoph, Julia </h3>
+
Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br>
-
<br>*cloning His_ & Strep_ Dig_Split_FokA into pJS 419 phagemid vector
+
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br>
-
- digested pEX vectors & pJS419 w/ XbaI, NotI using 16 µL DNA<br>
+
Buffer: 10 µl, QuickLigase Buffer NEB<br>
-
--> 1 % agarose gel
+
QuickLigase: 1 µl<br><br>
-
<br>*ssDNA production
+
-
- digestion of pET-39b(+): EcoRI, XbaI<br>
+
-
- digestion of M13 dsDNA: BspTI (isoschizomer of AflII), NarI<br>
+
-
<br>* Miniprep. of pET-39b+ ,stored in -20°C
+
-
<br>* Miniprep. of RV_Test ,stored in -20°C
+
-
<br>* phage display
+
-
- error prone PCR<br>
+
-
50µl PCR buffer (10x) without MgCl2<br>
+
-
50µl MnCl2 (5mM)<br>
+
-
120µl MgCl2 (25mM)<br>
+
-
15µl primer #7<br>
+
-
11,7µl primer #1<br>
+
-
20µl dNTPs (each 10mM)<br>
+
-
10µl Taq<br>
+
-
218,3µl water<br>
+
-
apportion total into 2 tubes à 245µl<br>
+
-
add 5 µl of plasmid 425-2 and 428-6 (each 50ng/µl)<br>
+
-
make aliquots à 50µl<br>
+
-
- ligation<br>
+
Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI<br>
-
2 samples:<br>
+
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br>
-
2µl vector<br>
+
Buffer: 10 µl, QuickLigase Buffer NEB<br>
-
12µl insert (digest 87/88)<br>
+
QuickLigase: 1 µl<br><br>
-
1µl Ligase<br>
+
 
-
2µl Ligase buffer<br>
+
Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br>
-
3µl water<br>
+
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI<br>
 +
Buffer: 10 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI<br>
 +
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI<br>
 +
Buffer: 10 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI<br>
 +
Buffer: 10 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI<br>
 +
Buffer: 10 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 6 µl Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI<br>
 +
Buffer: 10 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI<br>
 +
Buffer: 14 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI<br>
 +
Buffer: 14 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI<br>
 +
Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI<br>
 +
Buffer: 14 µl, QuickLigase Buffer NEB<br>
 +
QuickLigase: 1 µl<br><br>
 +
 
 +
<br>*did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
 +
 
 +
 
 +
<br>*ELISA
 +
-Blocking<br>
 +
-Washing with TBST-EDTA 5x<br>
 +
-Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM biotin-target DNA<br>
 +
-Incubation at 20°C in shaking<br>
 +
-Incubatet Phages with Oligo for 30min at 55°c waterbath<br>
 +
-Washing of wells and immunotubes with TBST-EDTA 5x<br>
 +
-Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1 phage+Oligo and 1+control as a control<br>
 +
-Incubation in shaker at 20°C<br>
 +
-Loaded immunotubes (each 1.5 ml) with 50µl P1, 25µl 449Phage+ Oligo in TBST-EDTA<br>
 +
-Incubation in shaker at 20°C<br>
 +
-Washing of wells and immunotubes with TBST-EDTA 5x
 +
-Loaded 100µl/well anti-M13 ab(1:1000), incubated for 1 hour at 20°C in shaker<br>
 +
-Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS 3x<br>
 +
-2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS-T 3x<br>
 +
-3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis<br>
 +
 
 +
<br>*Made new "AGO BB PCR" this time just using the ad-on-tail-primer for the bb pre- and suffix, so that hte EcoRI site should remain. Still the same not fitting bands were obtained-> the problem might be caused by multimerising of the primers, even so we were not able to predict any of tose using vectir nti or compairable programms...
 +
<br>* AGO assay using different targets-> were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration
 +
<br>* Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS
 +
<br>* Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I
 +
<br>* Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence
-
3 samples:<br>
 
-
2µl vector<br>
 
-
20,4µl insert (digest 87/89)<br>
 
-
1µl Ligase<br>
 
-
2,6µl Ligase buffer<br>
 
-
<br>*digest of pExStrepDigSplitFoka from 29.08.09 clon 1b because ligation or trafo didn't work and concentration of insert after gelex was probably too low
 
-
Plasmid: 10 µl pExStrepDigSplitFoka, clone from 29.08.09<br>
 
-
water: 14.5 µl<br>
 
-
BSA: 0.5 µl NEB iGEM stock<br>
 
-
buffer: 3 µl buffer 3 NEB iGEM stock<br>
 
-
Restriction_enzyme_1 : 2 µl XbaI from NEB KuKlabstock<br>
 
-
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock <br>
 
-
->put in 37°C for 2h
 
-
<br>*preparative gel of digest -> didn't work (no insert could be seen)
 
-
<br>*sequence of pExHisFluASplitFoki from 26.08.09, clone 100ul checked: ok
 
-
<br>*protein expression of pEx_HisFluASplitFoki in BL21de3 --> pellets frozen at -80°C
 
-
<br>*phosphorylised AGO A1 guide oligo via T4 Polynucleotid Kinase (with T4 Ligase Buffer) Final 20µl with 2.5 µM Oligo
 
-
<br>*Started making Phages with wt Aa AGO: DsbA-flag-AGO-CDg3p; DsbA-flag-AGO-noAmber-CDg3p; TorA-flag-AGO-CDg3p; TorA-flag-AGO-noAmber-CDg3p
 
-
->  Cells with the DsbA Phagmid-vektor showed very poor growth
 
-
<br>* Prepared Phage ELISA via Anti-Flag as well as Strep-Biotinylated target Oligo
 
-
<br>
 
</div>
</div>
<div style="text-align: left;"><br />
<div style="text-align: left;"><br />

Revision as of 21:30, 13 October 2009

FREiGEM 2009

October


01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel


* Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
* Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
* plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
* plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1
* glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80°C

* protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
* SDS gel of protein purification HisFluASplitFoki [[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel; HisFluASplitFoki; Lanes: NEB prestained protein marker, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, flow through fraction 2, washing fraction 2|400x400px]]
* pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
*phage display: - desalt ligation products (for electroporation)
pool samples (~80µl 445+87/89 and ~52µl 445+87/88)
add 1 volume of isopropanol, mix
-80°C for 10 minutes
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant
add 1 volume of 75% ethanol (without mixing)
centrifuge for 10 minutes at max rpm, 4°C; discard supernatant
let dry on heat block (50°C), lid open
add 25µl water
thermo shaker for 1h, 45°C, 1000rpm - PCR (4 samples each template):
buffer (with MgCl2): 5µl
primer #7: 1,5µl
primer #1: 1,17µl
dNTPs: 2µl
Taq: 1µl
MnCl2: 0,5µl
MgCl2: 5µl
water: 33µl
DNA (425 or 428): 5µl
- PCR:
DNA templates: pJs#448 (0,3µl), pJs#449 (0,3µl), pJs#375 (1µl), pJs#413 (0,3µl), pJs#445 (0,3µl)
primers (#95, #7): 1,5µl
high fidelity buffer: 5µl
dNTPs: 1µl
TMenzyme: 0,3µl
water: 39,7µl

* Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
*analysis of sequences from 28.09.09
*digest of pExStrepDigSplitFoka prep from 30.09.09 Plasmid: 15 µl
water: 9 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 3 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock

*digest of PCR product RBSStrepDigSplitFoka from 30.09.09 Plasmid: 10 µl
water: 14 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 3 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl PstI from NEB KuKlabstock

*digest of pExHisFluaSplitFoki prep from 24.08.09 Plasmid: 15 µl
water: 9 µl
BSA: 0.5 µl NEB iGEM stock
buffer: 3 µl buffer 2 NEB iGEM stock
Restriction_enzyme_1 : 1,5 µl XbaI from NEB KuKlabstock
Restriction_enzyme_2 : 1 µl SpeI from NEB KuKlabstock
-> put in 37°C room for 2h ->preparative gel
[[Image:Freiburg09_011009_fokverdaue.JPG|none|thumb|Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka || 400x400px]] interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration
->gelextraction
ligation of
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)

*Inoculation of - pJS419_StrepDigSplitFoka
- pJS419_HisDigSplitFoka
- pEx_HisFluA
- pEx_HisDigMiddleLiFoka

*digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
*Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09 --> make glycerol stocks tomorrow
*agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
*cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more)
*gel slices stored at -4°C over night

02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max


* phage display - OD600 of the pre-culture should be 0,2
- 37°C shaker
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)
- Apportion culture into 10x50 ml Falkon tubes
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)
- resuspend pellets in 25 ml H2O (10-> 8 tubes)
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4 tubes)
- 15 minutes on ice
- Centrifuge 10 min at 4°C / 2500
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-> 2 tubes)
- 5 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)
- 5 minutes on ice
- Centrifuge 10 min at 4°C / 2500 g
- Discard supernatant, pool pellets in 1 ml 10% DMSO
- Measure OD600 (1:100 dilution), OD should be 0,4
- Make aliquots à 80 µl, souse with N2
- Store at -80°C

*glycerol stock of pEx_HisFluASplitFoki in BL21de3
*SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09 --> do a Western Blot
*new ampicilin 100 aliquots
*preparation of M13dsDNA
*chem. competent cells aliqots
*Periplasma Project Digestion:
- digested with xba and spe --> GelBilder
* Gelextraction Min Elute Gel Extraction Kit
- mesure mass: StrepFokA = 50 mg - JS418= 90mg
-JS419=80mg
- HisFokA = 250mg
Nandropdata

*Ligation - with Quickligase JS119-StrepFokA
JS118-Strep
JS118-HisFokA
JS19-HisFokA
- 15 min at 25°C
*Transformation - 2YT-Medium 950 µL to 50µl cells and DNA
BL21-JS190
BL21-JS119StrepFokA
BL21-JS118Strep
BL21-JS118HisFokA
BL21-JS19HisFokA - Plated on KM plats

* Preparation of overnight cultures 5 ml LB medium each - pBad+Kan
-pET39B(+)
*Amp
--> Stored in 37°C room
*Digest: - pMASplitFoka
- pMAFoka
- pMAFoki
- pMASplitFoki
- pMALongLiFoka
- pMALongliFoki
- pMAShortli
- pMAMiddleli
- pMALongli
- pExStrepFluA

*preparative gel [[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA<|400x400px]] ->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly
*gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09
* digest and ligation into new pMA:
-pMASplitFokA clone1 19.08.09
-pMAFokA clone2 05.08.09
-pMAFoki clone2 05.08.09
-pMASplitFoki clone2 06.08.09
-pMAshortLi clone2 10.09.09
-pMAmiddelLi clone2 10.09.09
-pMAlongLi clone1 10.09.09
-pMAlongLiFokA clone1 10.09.09
-pMAlongLiFoki clone1 10.09.09
digestion with EcoRI and SpeI (vector and insert)

* Transformation of:
-pMAFokA clone2 05.08.09
-pMAFoki clone2 05.08.09
-pMASplitFoki clone2 06.08.09

*plasmidprep of:
1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick
2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick
3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick

4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick
5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick
6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick

7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit1
8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit2
9. pEX_DsbA+Strep+Dig+Split+FokA Clone3

10. pEX_DsbA+His+Dig+Split+FokA Clone1 --> seq 02.10.09 Gerrit3
11. pEX_DsbA+His+Dig+Split+FokA Clone2 --> seq 02.10.09 Gerrit4
12. pEX_DsbA+His+Dig+Split+FokA Clone3


*testdigest of: V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick
V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick
V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick

V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick
V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick
V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick

V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1
V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2
V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3

V10. pEX_DsbA+His+Dig+Split+FokA Clone1
V11. pEX_DsbA+His+Dig+Split+FokA Clone2
V12. pEX_DsbA+His+Dig+Split+FokA Clone3

V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)
V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)
with NcoI-HF and XbaI 1h at 37°C
--> geldbild

03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph


*plasmid prep of pET and pBad and Glytsocks(Xbl)
*new aliquots Ampicilin (70%EtOH)
*Test digest M13dsDNA+fokI as control for dsDNA , the pictures showed the two expected lanes(hardly visible on the printout). [[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel; M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09, M13dsDNA02.10.09 digest fokI |400x400px]]
*digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)
- pEx-Strep-Dig-MiddleLinker-Fok(inactive)
- pEx-Strep-Dig-ShortLinker-Fok(inactive)
- pEx-Strep-Dig-SplitLinker-Fok(inactive)
- pEx-His-Dig-SplitLinker_Fok(inactive)
- pEx-His-Dig-SplitLinker_Fok(active)
- pEx-Strep-Dig-SplitLinker-Fok(active)
- pEx-His-Dig-LongLinker-Fok(inactive)
- pEx-His-FluA-LongLinker-Fok(inactive)
- pEx-His-FluA-MiddleLinker-Fok(inactive)
- pEx-His-FluA-ShortLinker-Fok(inactive)
- pEx-His-FluA-SplitLinker-Fok(inactive)
- pEx-His-Dig
- pMA
with NgoMIV and SpeI
*digest of - pMA
with XbaI and AgeI
*Ligation of: - Strep-Dig-LongLinker-Fok(inactive)
- Strep-Dig-MiddleLinker-Fok(inactive)
- trep-Dig-ShortLinker-Fok(inactive)
- Strep-Dig-SplitLinker-Fok(inactive)
- His-Dig-SplitLinker_Fok(inactive)
- His-Dig-SplitLinker_Fok(active)
- Strep-Dig-SplitLinker-Fok(active)
- His-Dig-LongLinker-Fok(inactive)
- His-FluA-LongLinker-Fok(inactive)
- His-FluA-MiddleLinker-Fok(inactive)
- His-FluA-ShortLinker-Fok(inactive)
- His-FluA-SplitLinker-Fok(inactive)
into pMA
and
- Strep-Dig-LongLinker-Fok(inactive)
- Strep-Dig-MiddleLinker-Fok(inactive)
- trep-Dig-ShortLinker-Fok(inactive)
into new pEX
*inoculation of: -pMASplitFokA clone1 19.08.09
-pMAFokA clone2 05.08.09 (new)
-pMAFoki clone2 05.08.09 (new)
-pMASplitFoki clone2 06.08.09 (new)
-pMAshortLi clone2 10.09.09
-pMAmiddelLi clone2 10.09.09
-pMAlongLi clone1 10.09.09
-pMAlongLiFokA clone1 10.09.09
-pMAlongLiFoki clone1 10.09.09

-pMAFokA clone2 05.08.09 (old)
-pMAFoki clone2 05.08.09 (old)
-pMASplitFoki clone2 06.08.09 (old)

*inoculation and plasmid preperation: -pEX_strepFluA
*Quenching test with HisFluASplitFoki and fluorescin-tagged oligos
*BB Ago Pcr via Taq
*Started makin new VCS M13 Phages see Protocol day 1

Plan for 04.10.09


*inoculation of - pMA-Strep-Dig-LongLinker-Fok(inactive)
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)
- pMA-Strep-Dig-ShortLinker-Fok(inactive)
- pMA-Strep-Dig-SplitLinker-Fok(inactive)
- pMA-His-Dig-SplitLinker_Fok(inactive)
- pMA-His-Dig-SplitLinker_Fok(inactive)
- pMA-Strep-Dig-SplitLinker-Fok(active)
- pMA-His-Dig-LongLinker-Fok(inactive)
- pMA-His-FluA-LongLinker-Fok(inactive)
- pMA-His-FluA-MiddleLinker-Fok(inactive)
- pMA-His-FluA-ShortLinker-Fok(inactive)
- pMA-His-FluA-SplitLinker-Fok(inactive)
into LB-Amp
*digestion of pEx-Strep-Flua with AgeI and PstI
*digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive)
*ligation of the above parts and transformation into RV308
*plasmid preparation of... -pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLi
-pMAmiddelLi
-pMAlongLi
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)

04.10.09, Laura, Anika, Max


* Plasmid preparation of -pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 1 and 2,respectively - results see notebook

* Glycerolstock of
-pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 3
* Pellets of
-pMASplitFokA
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 1 and 2,respectively - stored in Pelletbox, -20°C
*Digest of 1. pEXHisDigMiddleLFoki 2. pMaScFaantiNIP 3. prep of Ligation pEX+CAT of 09.07.09 old 4. pMAShortLFoka
5. pMAShortLFoki
6. pMAMiddleLFoka
7. pMAMiddleLFoki
8. pMALongLFoka
9. pMALongLFoki
10.pEXStrepFlua

*Preparative Gel of Digest
[[Image:Freiburg09_041009_umklonierung_gel.JPG|none|thumb|Agarose gel; Lanes: 1. pEXHisDigMiddleLFoki, 2. pMaScFaantiNIP, 3. pEX+CAT, 4. pMAShortLFoka, 5. pMAShortLFoki, 6. pMAMiddleLFoka, 7. pMAMiddleLFoki, 8. pMALongLFoka, 9. pMALongLFoki,10.pEXStrepFlua|400x400px]] Freiburg09_041009_umklonierung_gel.JPG
*Gelextraction

*Ligation of
1.pMAHisDigMiddleFoki
2.pMACAT
3.pEXStrepFluAShortLFokA
4.pEXStrepFluAShortLFoki
5.pEXStrepFluAmiddleLFokA
6.pEXStrepFluAMiddleLFoki
7.pEXStrepFluALongLFokA
8.pEXStrepFluALongLFoki
9.JS 419StrepDigSplitFokA
10.JS 419HisDigSplitFokA
11.JS 418HisDigSplitFokA
Approach: 8µl H2O, 3 µl Vector, 6µl Insert, 2µl Quick Ligase Buffer, 1 µl Quick Ligase, 15 min, Room Temp.

*Dephosphorylation of pBAD Vector:
Approach: 2µl Eluat, 3,1 µl Fast Ap Buffer 10x, 1µl Fast Ap, 10 min. 37°C, 5 min. 75°C

*Transformation of
1.pMAHisDigMiddleFoki (RV)
2.pMACAT(RV)
3.pEXStrepFluAShortLFokA(RV)
4.pEXStrepFluAShortLFoki(RV)
5.pEXStrepFluAmiddleLFokA(RV)
6.pEXStrepFluAMiddleLFoki(RV)
7.pEXStrepFluALongLFokA(RV)
8.pEXStrepFluALongLFoki(RV)
9.pBAD (RV)
10.pJS 419StrepDigSplitFokA(XBL)
11.pJS 419HisDigSplitFokA(XBL)
12.pJS 418HisDigSplitFokA(XBL)

*test digest of plasmid preps from today -> run on a gel with digest pET39, xbaI, ecoRI from Tobi
*starter culture of pEx_DsbA_HisDigSplitFoka -> modified expression has to be done tomorrow
*inoculation of pJS418 and pJS419 for glycerolstocks tomorrow
*Taq AGO BB PCR did not work
*Phageproduction day 2

05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika


* 3l LB and 2L LB Agar
*Periplasma Project
*digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09)
*test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka
*1) Gelextraction
--> Gelbild
1) Vector: pEXHisFluSplitFoki
2)Insert: RBSStrepDigSplitFokA
3)PET39b+

*2)Ligation
-10ul 2 fold buffer
- 6 µl Insert
-3µl Vector
-1µl Quick Ligase
--> 15 min at RT
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA

*3)Transformation
-5 µl DNA of Ligation + BL21d3 and XBL
*'''Mini prep with with Qiagen Spin Miniprep Kit of''' - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3
- pMA-Dbsa 1,2,3
*'''Made Glycerolstocks of'''
*700µl Cellsuspension+ 300µl Glycerol - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3
- pMA-His-Dig-SplitLinker_Fok(active)1,2,3
- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3
- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3
- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3
- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3
- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3
- pMA-Dbsa 1,2,3
*'''Test Digestion of'''
*put in each sample: 5µl DNA, 2,5µl H2O, 1µl Buffer 3, 1µl EcoRI, 1µl PstI, 1µl BSA - pMA-Strep-Dig-LongLinker-Fok(inactive)3
- pMA-Strep-Dig-MiddleLinker-Fok(inactive)1
- pMA-Strep-Dig-ShortLinker-Fok(inactive)1
- pMA-Strep-Dig-SplitLinker-Fok(inactive)3
- pMA-His-Dig-SplitLinker_Fok(inactive)1
- pMA-His-Dig-SplitLinker_Fok(active)2
- pMA-Strep-Dig-SplitLinker-Fok(active)1
- pMA-His-Dig-LongLinker-Fok(inactive)1
- pMA-His-FluA-LongLinker-Fok(inactive)3
- pMA-His-FluA-MiddleLinker-Fok(inactive)3
- pMA-His-FluA-ShortLinker-Fok(inactive)1
- pMA-His-FluA-SplitLinker-Fok(inactive)2
- pMA-Dbsa 2

* Transformation of
- XBL pEXHisFluASplitFokIRBSStrepDigSplitFokA
- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA

*Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each - induced with 0,7mM IPTG and took samples T0-T5
- centrifuged in buckets 17', 4000rpm, 4°C
- eluted in 20 ml TES in each bucket
....
*PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones
*PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature

06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika


*digest - pBAD vector
DNA: 16.8µl
enzyme: 1µl AgeI
buffer: 2µl buffer 1
- pBAD insert
DNA: 16.8µl
enzyme: 1µl XmaI
buffer: 2µl buffer 4
BSA (10X): 2µl
- pJS 418/419
DNA: 10µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 3µl buffer 3
BSA (10X): 3µl
- pExStrepDigSplitFoka/pExHisDigSplitFoka
DNA: 16.8µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 3µl buffer 3
BSA (10X): 3µl

*testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: 5µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 1µl buffer 3
BSA (10X): 1µl

*preparative gels of digests
[[Image:Freiguburg09_061009_bad_js.JPG|none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418|300x300px]] [[Image:Freiburg09_061009_js_foka.JPG |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
interpretation: pExStrepDigSplitFoka seemed to be a wrong construct, the other constructs showed bands of the right size even if the concentration of the PCR pBAD insert seems to be very low
*testdigest of pMAdsba DNA: 5µl
enzyme: 1µl PstI and 1.5 µl Xba/
buffer: 1µl buffer 3
BSA (10X): 1µl

*His-tag purification of HisDigSplitFoka (periplasm export) with Ni-NTA column. Washing buffer: 25mM imidazole -cells were sonicated for 2 x 1min before filtering with 0.45µm and 0.22µm filter
*Ligation - Dephphorylation of pBAD Gelex
- 1µl Fast AP
- 5.5µl fast AP buffer
-1.5µl water
--> Solution was given to eluat - for each ligation:
- 6µl Insert
-3µl Vetor
-1µl Quickligase
-10µl buffer
--> pBAD
* Insert (Dummy)
--> pJS419+HisDigSplitFoka -->pJ418+HisDigSplitFoka
* Transformation --> pBAD
* Insert (Dummy)
--> pJS419+HisDigSplitFoka -->pJ418+HisDigSplitFoka - in XLblue
-pBAD was plated on AMP plates
- Both pJS... were plated on CM plates

*'''Ligation and Transformation of''' Ligation:per sample 8µl H2O; 3µl vector; 6µl insert; 2µl Quick Ligase buffer; 1µl Quick Ligase Transformation:Defrost competent cells on ice:(100 µl); add of the ligation: 5 µl; DNA and cells: mix softly by knocking; Incubation on ice for: 20-30 min; Heat shock at: 42°C for 40 sec; Cool off on ice for: 5 min; add sterile LB(or dyt)medium: 900 µl; Incubation in(shaker)at: 37°C for 60-70 min; Plate cells on LB+antibiotic plates: ampicillin; ligation: 2 plates: 1. 50µl cells 2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out: -pMA HisDig Middle Linker Foki
-pMA CAT
-pEX Strep FluA SL FokA
-pEX Strep FluA LL Foki
-pEX Strep FluA ML Foki
-pEX Strep FluA ML Foka
-pEX Strep FluA LL Foka

*Sequencing: 27µl H2O; 3µl DNA -pMA Dbsa clone 2
-pMA HisDigSplitFokA clone 2
-pMA HisFluASL Foki clone 1
-pMA HisFluASplitFoki clone 2
-pMA StrepDigLLFoki clone 3
-pMA HisFluaMLFoki clone 3
-pMA StrepDigSplitFokA clone 1
-pMA StrepDigMLFoki clone 1
-pMA StrepDigSLFoki clone 2
-pMA HisDigLLFoki clone 1
-pMA StrepDigSplitFoki clone 3
-pMA HisDigSplitFoki clone 1
-pMA HisFluaLLFoki clone 3

*Inoculation of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight

*Yesterdays improved pcr resulted in a lot of product, but still unspecific ones.. made new one with even higher annealing temperature (69°C) and a bit less cycles (35)
*New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 and A4 _see gele_ no differend results were obtained compaired to the assays using unphorphorylated oligos
*Started dialysis to transfer the leftover AGO-proteins into the assay buffer

07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika


*SDS gel of protein purification of HisDigSplitFoka (periplasm) from 06.10.09 [[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker broad range, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution fraction 6, flow through fraction 3, washing fraction 2, periplasm extract (frozen over night at -80°C)|400x400px]]
* Inoculation 4 Clones respectively:
- pMA-His-Dig-MiddleLinker-Foki in XLBlue
- pJS419-HIs-Dig-Split-Foka- in XLBlue
- pJS418-HIs-DIg-Split-Foka- in XLBlue
- pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest
- pEX-Strep-FluA-LongLinker-Foki in XL1blue rest
- pMA-CAT in XLblue 10µl
-pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest
2 Clones respectively:
-pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09 - pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09
* Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3
*two step PCR assembly of DsbA, His_Fos, and SplitFoka - program name: Assembly
- three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step
*preparative gel of the PCR samples -> primer haven't been diluted an probably made all secondary structures, has to be repeated
*digest of pBAD with AgeI and new PCR with insert digested with XmaI
*preparative gel with digest and
*test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka DNA: 5µl
Enzymes: 0.5µl of BamHI and MfeI each
Buffer: 1µl buffer 4
BSA (10fold): 1µl
Water: 2µl
-> pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1

*plasmidpreparation of - pEXHisFluASplitFoki
- pExStrepDigSplitFoka
-> low concentrations

*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl
Enzymes: 1µl of PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 10µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl

* Gelextraction of digest 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst)
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)

*preparative gels of digests
[[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]] [[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]
RBS PCR Product: m= 90 mg
vector: 0 0 130 mg

* Ligation - 6µl Insert
-3µl Vetor
-1µl Quickligase
-10µl buffer
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

*Transformation - All in XL1blue
- Vector: pEX-strep-Duig-Split-Foka
- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

*Stardet production of Phages baering the phagmid vektors with pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml each. see phageproduction protocoll day 1
*prepaired ELISA with anti-flag antibodies
*made electro competent cells for transformation with the ago phagmidbibliothek

08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes


*Poured SDS-Gels

*Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet
-Transfomation with 3µl M13 Phage stock (-80°) at 10:30Uhr , incubated for 4-5 hours at 37°C in shaker
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4°C room

*Miniprep - 2 clones respectively:
-A = pEX strep dig split foka
*Finished Phage production(see protocol day 2): We obtained approximately 3.8
*10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1
*10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of them to the anti-Flag ELISA

*Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):
see 405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to positive control (G5-6) and negative control (G3-4 and D3-10) detected about half an hour after the ABTS substrat was addet. <> 1 2 3 4 5 6 7 8 9 10 11 12
A 0.0470 0.0420 0.0440 0.0430 0.0480 0.0510 0.0460 0.0470 0.0470 0.0480 0.0450 0.0460
B 0.0470 0.0460 0.0480 0.0480 0.0460 0.0450 0.0470 0.0560 0.0470 0.0450 0.0450 0.0440
C 0.0460 0.0470 0.0500 0.0450 0.0430 0.0460 0.0450 0.0460 0.0430 0.0420 0.0440 0.0470
D 0.0490 0.0470 0.1590 0.1610 0.1920 0.1720 0.2210 0.2500 0.2130 0.2680 0.0460 0.0480
E 0.0490 0.0490 0.0450 0.0490 0.0430 0.0490 0.0460 0.0430 0.0460 0.0420 0.0450 0.0440
F 0.0490 0.0430 0.0470 0.0440 0.0470 0.0470 0.0440 0.0450 0.0500 0.0460 0.0450 0.0450
G 0.0500 0.0440 0.2530 0.2510 3.9180 3.8800 0.9560 0.9380 1.2420 1.2360 0.0440 0.0460
H 0.0480 0.0430 0.0510 0.0470 0.0480 0.0460 0.0470 0.0450 0.0450 0.0440 0.0460 0.0460

* Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction
*digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl
Enzymes: 1µl of PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExHisDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl
RBSStrepDigSplitFoka: 5µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 19µl
->made two digest of PCR construct

* Gelextraction of digest 1) Insert: His-Dig-Split-Foka( digested with xba and pst)
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)
and also PCR purification of RBS-Strep-Dig-Split-FokA
*preparative gels of digests ->see picture

*ligation of -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)
-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)

*transformation of ligations
*inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol
*new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel ->interpretation: no band of 921bp was visible

*one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel ->see picture
->interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible

*new one step PCR of pMADsba with newly prepared 1:1000 dilution
*test digest of plasmidpreparations from today ->see picture

09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika


*Phage ss DNA
-Over night in 4°C room precipitated phages: centrifuged for 20 min at 5000rpm and 4°C
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet recognized)
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at 13000rpm
-Decant the supernatant into new eppis, precipitate with 170µl PEG/NaCl and leave for 1 hour on ice

*Miniprep - pJS 419-his-dig-split-foka (clon 1+2)
- Glycerolstock of clon1 and 2
- 300µl Glycerol
- 700 µl culture
- Pellets from clones 3,5,6
Nanodrop data:
pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl
pJS 419-his-dig-split-foka-clon2 =468 ng/µl

*Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
*send to sequencing:
pMA Dig Plasmidprep 22.07.09 Timo1
pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2
pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3
pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4
pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5
pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6
pMA Cat plasmidprep clone1 08.10.09 Timo7
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9
pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12

* concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation
* electrical trafo of the 449 ago phagmid bibliothek into XL1
* prepaired immutubes with streptavidin for ago phages test panning tomorrow

10.10.09 Julia, Caro, Laura, Christoph


*Measured OD600 of over night ER2738 culture abs.1)0.218 (1:10);2)0.23 (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet

*Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM
*Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer flasks and infected with 15µl M13 phage, shake for 4 hours at 37°C
*Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4°C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl
*Precipitaion over night in 4°C room
*plasmidpreparation of - pBADQuick clones 1-6
- pBADT4 clones 1-6
- pMAYFP clone 1-2
- pMACFP clone 1
- pMASplitlinker clones 1-2
- HisTag clones 1-2 (but test digest was negative -> thrown away)*digest for recloning of pMA constructs: pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from 10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09, pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl
Enzymes: 1µl of NgoMIVI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl
pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from 25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from 01.10.09: 10µl each
Enzymes: 1µl of AgeI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl

*PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together
*analytical gel of PCRs -> didn't work
*preparative gels of digests -> see picture -> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more
*ligation of -pMAStrepDigShortLiFoka
-pMAStrepDigMiddleLiFoka
-pMAStrepDigLongLiFoka
-pExStrepDigShortLiFoka
-pExStrepDigMiddleLiFoka
-pExStrepDigLongLiFoka
-pMAHisDigShortLiFoka
-pMAHisDigMiddleLiFoka
-pMAHisDigLongLiFoka
-pMACATNd4 (MQI)
-pMACATNd4 (MQII)
-pMA Kontrolle (M)
-> in XLBlue
- pEx_CATNd4 (EQI)
- pEx_CATNd4 (EQII)
- pEx_Kontrolle(E)
->RV308 Insert: 6µl
vector: 3µl
Ouick ligase buffer: 10µl
Quick ligase: 1µl

*transformation of ligations in XBL on LB/Amp/1%glucose plates
*cotransformation of
*testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture
[[Image:Freiburg09 101009pet39b+ssdnaagoassay.JPG|none|thumb|gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA|400x400px]]
*cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates
*inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09

11.10.09, Timo, Hannes, Max, Anika


* Digestion of 1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI
2.pMAFos_HlsbZip, XbaI - AgI
3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI
4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI
5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI
6.pMA_BB057, XbaI - PstI (01.10.09)

*gel extraction of - pMA
- pEx
- ...
* Testdigestion of 7.pMA_YFP 2 (10.10.09, Caro)
8.pMA_CFP (10.10.09, Caro)
9.pMASplitLi1 (10.10.09, Caro)
10.pMASplitLi2 (10.10.09, Caro)
...image..
* 1% Agarosegel of constructs above
* Phage breeding day 2
* Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3)
* plasmid prep of pEx_DsbA_StrepDigSplitFoka
* plasmid prep of pEx_HisDigSplitFoka
* M13 ssDNA produced with bacterial of Julia and tried another variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected with
M13phage particles and let it grow for 2h. After this followed the qiagen M13 protocol for M13Dna

12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit


* protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22°C.
* made 5 litres of DYT
*digest of pMASplitFoka clone 1 and 2 from 04.10.09 DNA:10µl Enzymes: 1µl of NgoMIVI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (100fold): 0.5µl
Water: 14.5µl

* Plasmidprep. of:
1.pMAFokA (old)
2.pMA-CAT Ndelta4 (MQII)1
3.pMA-CAT Ndelta4 (MQII)2
4.pMA-CAT Ndelta4 (MQII)3
5.pMA-CAT Ndelta4 (MQII)4
6.pMA-CAT Ndelta4 (MQII)5
7.pMA-CAT Ndelta4 (MQII)6
8.pMAFoka clone 1 from 04.10.

*Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen...
*Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay
*Made digest of errorprone PCR product of the AGO-G3P constructs (from 02.10.; one with, one without Amber) via NheI and SfiI to gain new Phagmid library
*Send to sequencing:[[Protocols#DNA_Sequencing]]
Julia 1-6:
1)pBADQuick clone 3
2)pBAD T4 clone 2
3)pBAD T4 clone 1
4)pMA YFP clone 1
5)pMA CFP clone 1
6)pMA Split Linker clone 2

Gerrit1: pMA_cat-Nd4
*Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for Panning Simulation for tomorrow
*made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki ->will prepare electrocompetent cells tomorrow

*inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp
*PCR of pMADsba repeated with different (higher) template dilutions ->analytical gel (see picture)
->didn't work again

*poured IPTG/XGal plates for in vivo plaque assay test run
*transformation of:
- pBAD_FokA (Quick)
- pBAD_CAT(Quick)
- pBAD_FokA (T4)
- pBAD_CAT (T4)
- pEX_FokA+YFP
- pEX_DsbA+FokA+YFP
into BL21 de3 gold
Plates: LB+ AMP +1%Glucose

*cultivation of pma strep dig split foka
pma long linker
pma his flua split foki

13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia


*Miniprep -pMA-fos 1
-pMA-fos 2
-pMA his flua split fokI
- pMA strep dig split foka
-pMA long linker

*testdigest of Plasmidprep from today 15 µl for 6µl loading dye -buffer 2 1µl
-xbaI 0.5µl
-pstI 0.75µl
-DNA 2 µl
-water 9.75µl
-BSA 1µl
--> Gelbild - Glycerolstocks are in in box of 5/10/2009
*made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka) ->stored in -80°C
*culture of ER2738 in LB Tet for cotransformation in afternoon
*test in vivo assay - electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV
- 1.5h on 37°C shaker at 750rpm
- precipitation of phages
- infection of ER2738 with different dilutions of phages
- mixed cells with Top agar and plated them on IPTG/XGAL plates -> 37° shaker

*inoculation of -pMASplitFoka clone 1 from prep 04.10.09
-pMAStrepDig clone 2 from prep 25.09.09
-pMAMiddleFoka clone 2 from prep 04.10.09
-pMAShortFoka clone 2 from prep 04.10.09
-pMAFoka clone 1 from prep 04.10.09
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09
-pMALongFoka clone1 prep from 10.09.09
-pMAFos 13.10.09
-pBAD_CAT 13.10.09
-pBAD_Foka 13.10.09
-pMADsbAHisDigSplitFoka 13.10.09
-pMADsbAStrepDigSplitFoka 13.10.09
-pExFosSplitFoka 13.10.09
-pMAFosSplitFoka 13.10.09

*protein purification of DsbA_StrepDigSplitFoka with Strep column
*digestion, 1% agarose gel and gel extraction of - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI
- pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI => wasn't ok on the gel, inoculated clone 1
- pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
- pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
- pMA-His-Dig (24.08., clone 2) with AgeI + PstI
- pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
- pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI
- pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI
- pEx-Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI - pMA (01.10., c=500ng/µl) with XbaI + AgeI - pMA (01.10., c=500ng/µl) with EcoRI + SpeI
*ligations: Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI
Insert: 6 µl LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-His-Dig (24.08., clone 2) with AgeI + PstI
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
Insert: 6 µl ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
Insert: 6 µl MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 6 µl Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI
Buffer: 10 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl ShortLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl MiddleLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl

Vector: 3 µl pMA (01.10., c=500ng/µl) with XbaI + AgeI
Insert: 2 µl LongLinker, hybridised, with XbaI + AgeI
Buffer: 14 µl, QuickLigase Buffer NEB
QuickLigase: 1 µl


*did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
*ELISA -Blocking
-Washing with TBST-EDTA 5x
-Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM biotin-target DNA
-Incubation at 20°C in shaking
-Incubatet Phages with Oligo for 30min at 55°c waterbath
-Washing of wells and immunotubes with TBST-EDTA 5x
-Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1 phage+Oligo and 1+control as a control
-Incubation in shaker at 20°C
-Loaded immunotubes (each 1.5 ml) with 50µl P1, 25µl 449Phage+ Oligo in TBST-EDTA
-Incubation in shaker at 20°C
-Washing of wells and immunotubes with TBST-EDTA 5x -Loaded 100µl/well anti-M13 ab(1:1000), incubated for 1 hour at 20°C in shaker
-Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS 3x
-2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted supernatant into eppis, washed with TBS-T 3x
-3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis

*Made new "AGO BB PCR" this time just using the ad-on-tail-primer for the bb pre- and suffix, so that hte EcoRI site should remain. Still the same not fitting bands were obtained-> the problem might be caused by multimerising of the primers, even so we were not able to predict any of tose using vectir nti or compairable programms...
* AGO assay using different targets-> were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration
* Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS
* Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I
* Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence






Retrieved from "http://2009.igem.org/Team:Freiburg_bioware/Notebook/September"