Team:Freiburg bioware/Notebook/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)

•  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

• digest of PCR product RBSStrepDigSplitFoka from 30.09.09 Plasmid: 10 µl

Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka

• Inoculation of - pJS419_StrepDigSplitFoka

• 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

• 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)

• digest of - pMA with XbaI and AgeI

• Ligation of: - Strep-Dig-LongLinker-Fok(inactive)

• inoculation of: -pMASplitFokA clone1 19.08.09

• 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)

• 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

04.10.09, Laura, Anika, Max

•  Plasmid preparation of -pMASplitFokA

•  Glycerolstock of

•  Pellets of

• Digest of 1. pEXHisDigMiddleLFoki 2. pMaScFaantiNIP 3. prep of Ligation pEX+CAT of 09.07.09 old 4. pMAShortLFoka

• 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

• Dephosphorylation of pBAD Vector:

• Transformation of

• 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

• 2)Ligation

• 3)Transformation

• '''Mini prep with with Qiagen Spin Miniprep Kit of''' - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3

• Made Glycerolstocks of
• 700µl Cellsuspension+ 300µl Glycerol - pMA-Strep-Dig-LongLinker-Fok(inactive)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

•  Transformation of

• Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each - induced with 0,7mM IPTG and took samples T0-T5

• 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

• testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: 5µl

• 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

• 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

•  Insert (Dummy)

•  Transformation --> pBAD
•  Insert (Dummy)

• Ligation and Transformation of

• Sequencing: 27µl H2O; 3µl DNA -pMA Dbsa clone 2

• 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:

•  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

• plasmidpreparation of - pEXHisFluASplitFoki

• digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl

•  Gelextraction of digest 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst)

• 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]]

•  Ligation - 6µl Insert

• Transformation - All in XL1blue

• 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

• 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

•  Gelextraction of digest 1) Insert: His-Dig-Split-Foka( digested with xba and pst)

• preparative gels of digests ->see picture

• ligation of -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka

• 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

• Miniprep - pJS 419-his-dig-split-foka (clon 1+2)

• Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
• send to sequencing:

•  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

• 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

• 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

• 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 

• gel extraction of - pMA

•  Testdigestion of 

•  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

•  Plasmidprep. of:

• 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]]

• 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:

• cultivation of pma strep dig split foka

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

• Miniprep -pMA-fos 1

• testdigest of Plasmidprep from today 15 µl for 6µl loading dye -buffer 2 1µl

• 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

• inoculation of -pMASplitFoka clone 1 from prep 04.10.09

• 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

• ligations: Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI

• did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
• ELISA -Blocking

• 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