"
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)
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
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
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
 |
| Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka |
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
- 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
- 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:
- Gelextraction Min Elute Gel Extraction Kit
Ligation - with Quickligase JS119-StrepFokA
Transformation - 2YT-Medium 950 µL to 50µl cells and DNA
Preparation of overnight cultures 5 ml LB medium each - pBad+Kan
Amp
Digest: - pMASplitFoka
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:
Transformation of:
plasmidprep of:
testdigest of: V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick
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-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)
- 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
-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-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
-pMAFoki
-pMASplitFoki
-pMAshortLi
-pMAmiddelLi
-pMAlongLi
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
04.10.09, Laura, Anika, Max
- Plasmid preparation of -pMASplitFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 1 and 2,respectively - results see notebook
- Glycerolstock of
-pMAFokA
-pMAFoki
-pMASplitFoki
-pMAshortLiFokA
-pMAshortLiFoki
-pMAmiddelLiFokA
-pMAmiddelLiFoki
-pMAlongLiFokA
-pMAlongLiFoki
-pMAFokA (old)
-pMAFoki (old)
-pMASplitFoki (old)
clone 3
- Pellets of
-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
6. pMAMiddleLFoka
7. pMAMiddleLFoki
8. pMALongLFoka
9. pMALongLFoki
10.pEXStrepFlua
- Preparative Gel of Digest
 |
| Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA |
- Gelextraction
- Ligation of
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:
- Transformation of
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
1) Vector: pEXHisFluSplitFoki
2)Insert: RBSStrepDigSplitFokA
3)PET39b+
- 2)Ligation
- 6 µl Insert
-3µl Vector
-1µl Quick Ligase
--> 15 min at RT
Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA
- 3)Transformation
- '''Mini prep with with Qiagen Spin Miniprep Kit of''' - pMA-Strep-Dig-LongLinker-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-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-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
- 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
- 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
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
buffer: 1µl buffer 3
BSA (10X): 1µl
- preparative gels of digests
 |
 |
| preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418 | preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |
- testdigest of pMAdsba DNA: 5µl
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
- 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)
- Transformation --> pBAD
- Insert (Dummy)
-pBAD was plated on AMP plates
- Both pJS... were plated on CM plates
- Ligation and Transformation of
-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 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:
- 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
- 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
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
-> low concentrations
- digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl
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)
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]] |
vector: 0 0 130 mg
- Ligation - 6µl Insert
-1µl Quickligase
-10µl buffer
pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka
- Transformation - All in XL1blue
- 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
-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:
- 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):
| 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
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)
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 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
- 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
-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)
- 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:
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
- 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
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
-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
 |
| gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA |
- 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
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
- ...
- Testdigestion of
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 withM13phage 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
BSA (100fold): 0.5µl
Water: 14.5µl
- Plasmidprep. of:
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]]
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)
- poured IPTG/XGal plates for in vivo plaque assay test run
- transformation of:
- 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 his flua split foki
13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia
- Miniprep -pMA-fos 1
-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
-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
- 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
-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-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
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
-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
