"
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
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
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
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
-
pJS419_HisDigSplitFoka
- pEx_HisFluA
- pEx_HisDigMiddleLiFoka
- 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
- 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
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-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
- 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
- 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)
-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
- 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)
-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
-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
-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
-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
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
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.
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)
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+
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
- 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
- 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
- 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
- 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
- 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
....
- 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
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
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 |
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
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
- 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
-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
-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
-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
Hybridize M13 ssDNA with Fok control oligos 2 and 3
| Volume | Reagent |
| 10 µl | M13 ssDNA (c = 127 ng/µl, 17.08.09, sample #5.2) |
| 5 µl | MgCl2 (5 mM) |
| 5 µl | Tris-HCl pH 8 (100 mM) |
| 2 µl | Fok control 2 (10 ng/µl; 1 µM) |
| 2 µl | Fok control 3 (10 ng/µl; 1 µM) |
| 30 µl |
The hybridization was also done with just Fok control 2 and with no oligos at all.
Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).
10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.
 Agarose gel of FokI cutting M13 ssDNA |
 Agarose gel of FokI cutting M13 ssDNA |
Lane 2: Untreated M13 ssDNA (250 ng);
Lane 3: Digest of M13 ssDNA with FokI and without oligo;
Lane 4: Digest of M13 ssDNA with FokI and with one oligo;
Lane 5: Digest of M13 ssDNA with FokI and two oligos.
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
- 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
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
-> 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
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)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)
- preparative gels of digests
 |
 |
| preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product | preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |
RBS PCR
Product: m= 90 mg
vector: 0 0 130 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
-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
- 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
-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
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
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)
-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
-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
- 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
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)
- 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
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
-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
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)
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..
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
Buffer:
3µl buffer 1
BSA (100fold): 0.5µl
Water: 14.5µl
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.
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
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
- 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
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
-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
-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
- 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
-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
- 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
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
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
14.10.09, Laura, Caro, Christoph, Hannes, Julia, Anika
- Made LB-medium, 0,7% LB-Agar, 50% Glucose * Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3
| OD measurements | 1 hour before | induction | 45min | 1.5h | 2h 45min |
| induction | |||||
| 1. clone1 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose | 0.1 | 0.74 | 1.19 | 1.25 | 2.34 |
| 2. clone1 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose | 0.1 | 0.67 | 1.08 | 0.94 | 2.20 |
| 3. clone 1 pBAD_CAT control | 0.07 | 0.72 | 1.08 | 1.24 | 2.23 |
| 4. clone2 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose | 0.07 | 0.63 | 0.96 | 1.11 | 2.00 |
| 5. clone2 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose | 0.07 | 0.66 | 1.06 | 1.25 | 2.21 |
| 6. clone2 pBAD_CAT control | 0.08 | 0.72 | 1.11 | 1.33 | 2.25 |
| 7. clone1 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose | 0.07 | 0.67 | 1.13 | 1.26 | 2.07 |
| 8. clone1 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose | 0.09 | 0.67 | 1.21 | 1.28 | 2.22 |
| 9. clone1 pBAD_Foka control | 0.09 | 0.7 | 1.11 | 1.38 | 2.31 |
| 10. clone2 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose | 0.07 | 0.6 | 0.93 | 1.16 | 1.87 |
| 11. clone2 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose | 0.07 | 0.62 | 0.94 | 1.18 | 1.93 |
| 12. clone2 pBAD_Foka control | 0.01 | 0.64 | 1.04 | 1.24 | 2.27 |
--> no
differences between induced and control cells
 |
 |
| SDS gel of testexpression with pBAD_CAT in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_CAT control, clone1 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_CAT control, clone2 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, NEB prestained protein marker borad range | SDS gel of testexpression with pBAD_Foka in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_Foka control, clone1 pBAD_Foka+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_Foka control, clone2 pBAD_Foka+0.6mM IPTG+0.002% Arabinose |
- SDS gel of DsbA_StrepDigSplitFoka purification from 13.10.09
 |
 |
| SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, elution fraction 8, elution fraction 9 | SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, flow through fraction 1, flow through fraction 2, flow through 3, flow through fraction 4, washing fraction 1, washing fraction 2, washing fraction 3, unpurified periplasm extract (frozen at -80°C) |
- Plasmidpreparation
1 =
pEX-fos-split-fokA-K1
2 = pEX-fos-split-fokA-K2
3 = pMA-DsbA-strep-dig-split-fokA-K1
4 = pMA-DsbA-strep-dig-split-fokA-K2
5 = pMA-DsbA-his-dig-split-fokA-K1
6 = pMA-DsbA-his-dig-split-fokA-K2
7 = pMA-fos-split-fokA-K1
8 = pMA-fos-split-fokA-K2
9 = pBAD-fokA-K1
10= pBAD-fokA-K2
11 = pBAD-CAT-K1
12= pBAD-CAT-K2
13= pMA-shortlinker-fokA
14= pMA-longlinkerFoka
15= pMA-strep-dig
16= pMA-middlelinker-fokA
18=pMA-split-fokA
->sequencing of clones 1 and 2 of pBAD_Foka
2 = pEX-fos-split-fokA-K2
3 = pMA-DsbA-strep-dig-split-fokA-K1
4 = pMA-DsbA-strep-dig-split-fokA-K2
5 = pMA-DsbA-his-dig-split-fokA-K1
6 = pMA-DsbA-his-dig-split-fokA-K2
7 = pMA-fos-split-fokA-K1
8 = pMA-fos-split-fokA-K2
9 = pBAD-fokA-K1
10= pBAD-fokA-K2
11 = pBAD-CAT-K1
12= pBAD-CAT-K2
13= pMA-shortlinker-fokA
14= pMA-longlinkerFoka
15= pMA-strep-dig
16= pMA-middlelinker-fokA
18=pMA-split-fokA
->sequencing of clones 1 and 2 of pBAD_Foka
- Pellets of:
19=pMA-fokA
20=pEX-DsbA-strep-dig-split-fokA
21=pEX-strep-fluA(3.10)
20=pEX-DsbA-strep-dig-split-fokA
21=pEX-strep-fluA(3.10)
- digest1: - pBAD-iGEM(Midi 11.10)
-
pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5)
- pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)
- pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)
-10µlDNA
-3µl buffer3
-0.5µlBSA(100fold)
-14.5µl water -1µl EcoRI
-1µl PstI
- loaded on 1% agagel
-3µl buffer3
-0.5µlBSA(100fold)
-14.5µl water -1µl EcoRI
-1µl PstI
- loaded on 1% agagel
- digest2: - pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)
-
pEX-fos-split-fokA-K1
-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14.5µl water -1µl xbaI
-1µl NgoNIV
-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14.5µl water -1µl xbaI
-1µl NgoNIV
loaded on 1% agagel
- digest3: -pMA-DsbA-clone2 (5.10)
-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14µl water -1µl xbaI
-1.5µl AgeI
loaded on 3% agagel *Gelextraction -pBAD iGEM
-DsbA-his-dig-split-foka
-DsbA-strep-dig-split-foka
-pMA-fos-dig-split-foka
-pEX-fos-split-foka
--> Picture
-3µl buffer4
-0.5µlBSA(100fold)
-14µl water -1µl xbaI
-1.5µl AgeI
loaded on 3% agagel *Gelextraction -pBAD iGEM
-DsbA-his-dig-split-foka
-DsbA-strep-dig-split-foka
-pMA-fos-dig-split-foka
-pEX-fos-split-foka
--> Picture
- Ligation -pBAD-DsbA-his-dig-split-fokA
-pBAD-DsbA-strep-dig-split-fokA
- pMA-fos-dig-split-fokA-DsbA
-pEX-fos-split-fokA-DsbA
-10µl Qick ligase buffer
-1 µl Quick ligase
-6µl Insert
-3µl Vector
- pMA-fos-dig-split-fokA-DsbA
-pEX-fos-split-fokA-DsbA
-10µl Qick ligase buffer
-1 µl Quick ligase
-6µl Insert
-3µl Vector
- Transformation of the ligation above in X1Blue competent cells
-100µl and rest
of each transformation were plated on
Amp/Gluc plates
- made top agar (0.7% agar)
- nothing grew on IPTG/Amp plates for test in vivo assay -> made control assays with
-
electroporating
cotransformed XL1Blue with ssM13DNA and plating them
directly to IPTG/Amp cells (no phage precipitation and infection of
ER2738) to test electrocompetence
- infection of ER2738 with phages from lab phage stock and plating them to IPTG/Amp cells to test plates
->put plates in 37° room overnight
- infection of ER2738 with phages from lab phage stock and plating them to IPTG/Amp cells to test plates
->put plates in 37° room overnight
- inoclutaion of ER2738 from lab stock and our stock in LB+Tet ->put them on shaker at 26°C
- XL1 was el. transformed with the ligated AGO-Phagmid library (errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence with and without amberstop from yesterday) and plated everything (4 ml)on two big tubs.
- new PCR for ssDNA from the digested pET39b+ vector to get new target for an additional AGO assay
- prepaired ELISA to repeat yesterdays AGO-Phage-ELISA tomorrow (because yesterdays results were pretty contradictory -> we probably made a mistake somewhere)
15.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph
- ELISA:
-Blocking:
350µl/well 1% BSA in TBST-EDTA, incubated at
20°C for 2h gently shaking
-5x washing with 200µl/well TBST-EDTA
-Incubation of phages with oligos for 30min in 55°C waterbath
-Incubation of 75µl/well phages for 1h at 20°C gently shaking
-5x washing with 200µl/well TBST-EDTA
-Antibody: add 75µl/well of anti- M13 antibody (1:1000)
-5x washing with 200µl/well TBST-EDTA
-Substrate: add 75µl/well 1mg/ml ABTS; measure absorbance immediately and after 3 and 5 min; 405nm
-5x washing with 200µl/well TBST-EDTA
-Incubation of phages with oligos for 30min in 55°C waterbath
-Incubation of 75µl/well phages for 1h at 20°C gently shaking
-5x washing with 200µl/well TBST-EDTA
-Antibody: add 75µl/well of anti- M13 antibody (1:1000)
-5x washing with 200µl/well TBST-EDTA
-Substrate: add 75µl/well 1mg/ml ABTS; measure absorbance immediately and after 3 and 5 min; 405nm
- made chemical competent XL1 Blue *test digests of plasmid preparations from 14.10.09 *seqencing of - laura1:pExFosSplitFoka clone 2
-
laura2:pMAFosSplitFoka clone 2
- laura3:pMADsbAHisDigSplitFoka clone 2
- laura4: pMADsbAStrepDigSplitFoka clone 1
- laura3:pMADsbAHisDigSplitFoka clone 2
- laura4: pMADsbAStrepDigSplitFoka clone 1
- inoculation of - ER2738 from our stock
- XL1 blue
from our stock
-> in 50 ml LB+Tetracyclin each, put in 37°C *inoculation of -pBAD_Foka clone 1 and 2 from 14.10.09
-pMAFoka clone 1from 04.10.09
-pDsbA clone 2 from 05.10.09
-> in 50 ml LB+Tetracyclin each, put in 37°C *inoculation of -pBAD_Foka clone 1 and 2 from 14.10.09
-pMAFoka clone 1from 04.10.09
-pDsbA clone 2 from 05.10.09
- for in vivo assay - measured pd.7.phage display library concentration new
- mixed
1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of
0.46) and XL1 blue (OD600 of 0.54) each
- let mixtures stand for 10min (infection time)
- mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal plates
- put it in 37°C roomin the dark
-pExStrepFluASplitFoki from 08.10.09
- let mixtures stand for 10min (infection time)
- mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal plates
- put it in 37°C roomin the dark
-pExStrepFluASplitFoki from 08.10.09
->
in 5 ml LB+Ampicillin each, put in 37°C
- new ssDNA production via pET39b+ digestion product one-primer-pcr
- elusions from the test panning was given to xl1-> infection-> plated in tet cm -> cells infected with the phagmid should grow->tomorrow picking clones and determination of the phagmidinsert
- new AGO BB pcr usind dmso, the gene cut out of the vektor and only the ad-on-tail-primers
- starter cultures of AGO Aa and AGO Tt in BL21de3 in DYT+Kan
- starter cultures of pEx-His-Fos-Split-Foka in RV308 in DYT+Amp+1% Glucose
16.10.09, Laura, Caro, Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph
- Made 10 L DYT
- Inoculation of elutions from panning: E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20
17.10.09, Laura, Julia, Manu, Hannes, Gerrit, Christoph
- Expression of Ago Tt in Bl21de3 OD measurement
| 1 hour before | ||||||
| induction | time of induction | 1.5 hours | 4 hours | 6.5 hours | 8 hours | |
| flask 1 | 0.29 | 0.53 | 0.86 | 1.18 | 1.32 | 1.51 |
| flask 2 | 0.31 | 0.57 | 0.91 | 1.21 | 1.38 | 1.49 |
| flask 3 | 0.32 | 0.51 | 0.9 | 1.31 | 1.32 | 1.53 |
| flask 4 | 0.28 | 0.49 | 0.88 | 1.15 | 1.33 | 1.57 |
| flask 5 | 0.27 | 0.51 | 0.89 | 1.26 | 1.32 | 1.48 |
| flask 6 | 0.32 | 0.51 | 0.83 | 1.24 | 1.5 | 1.61 |
- Dilution from XL1 from OD600 2,8 to OD600 0.07 *Minipreps from : E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20 wich are phagemids we cultured from elutions of panning from 13.10.09
- IPTG/X-Gal plates with infected ER2738 was blue, with the infected XL1 blue no blue plaques could be seen -> so infection by phages was positive and lots of blue plaques were built up
- made new in vivo test assay with cotransformated, electrocompetent XL1 blue - transformed M13 ss DNA from 17.08.09 into cells by electroporation at 1.7kV
- 1.5h
incubation time on 37°C shaker
- 80, 10 and 5µl of electroporated cells were mixed with 180µl XL1 blue and ER2738 with OD 600 of about 0.5
- incubation time of 10 min at room temperature
- mixing cells with 3ml topagar, vortexing shortly and plating on IPTG/X-Gal plates
- put in 37°C room overnight
- 80, 10 and 5µl of electroporated cells were mixed with 180µl XL1 blue and ER2738 with OD 600 of about 0.5
- incubation time of 10 min at room temperature
- mixing cells with 3ml topagar, vortexing shortly and plating on IPTG/X-Gal plates
- put in 37°C room overnight
- control of electrocompetence of XL1 blue by electroporation of pET28a with Kan resistance into XL1 blue and RV respecitvely -> comparison in the end or if colonies will show up at all will show if our cotransformated cells are competent at all *inoculation of - pMAFoka clone 1 from 04.10.09
- pMADsbA
clone 2 from 05.10.09
- pBADFoka clone 1 from 15.10.09
- pExStrepFluALongliFoki clone 1 from 08.10.09
- pBADFoka clone 1 from 15.10.09
- pExStrepFluALongliFoki clone 1 from 08.10.09
- miniprep of all clones picked from the elutions of the test panning to determine the efficiency of the selection
- new ssDNA via Pet39b+ one primer pcr
- new infection of XL1 cells with the elution from the test panning-> this time plated on lb agar with tet cm and GLUC (to suppress the expression of the phagmid insert)
18.10.09 Manu
- Expression of Ago Aa at 24°C *Expression of GST-FosW at 30°C
19.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph
- analysis of the geles of the digestion of the clones picked from the elution plates revealed not a single clone containing the ago insert-> possibly the lack of gluc on the plate led to a selection against this insert-> new klones picked from the plate with gluc
- new ago cleavage assay reveald last saturdays promising results as false. it seems like it was just two conformations of one ssDNA molecule
- starter cultures of pEx-His-Fos-Split-Fok_a
- protein purification of GST-FosW with Ni-NTA column - French pressed, centrifuged 15min at 20000rpm, sonicated and filtered
- column was washed with
25mM imidazole and eluted with 250mM imidazole
- in vivo assay retried with shorter incubation time
20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel, Max, Gerrit, Christoph, Caro, Hannes
- new round of expression with pEx-His-Fos-Split-Fok_a in BL21de3at 24°C
| OD measurement | time of | 2 hours | 3 hours | 3 hours | 4 hours |
| induction | 45 min | 15 min | |||
| flask 1 | 1.3 | 2.63 | 3.47 | 4.29 | 3.99 |
| flask 2 | 1.19 | 2.87 | 3.87 | 4.51 | 4.62 |
| flask 3 | 1.5 | 3.22 | 4.45 | 5.03 | 5.41 |
| flask 4 | 1.03 | 2.9 | 4.21 | 5.11 | 5.88 |
| flask 5 | 0.81 | 2.81 | 3.76 | 4.29 | 4.76 |
| flask 6 | 1.52 | 2.72 | 3.71 | 3.95 | 5.17 |
