Team:Hannover/Notebook
Notebook
| date | activity | summary | |
|---|---|---|---|
| 21.04.2016 | Bianca | prepared working area | pipets and other stuff autoklaved |
| 28.04.2016 | Janis, Bianca | test pipets | pipets work well |
| Assembly of TALEs | we cloned three different constructs in TALEs and transformed them into E.coli: 1) R-SCTALEN left HA Tag 2) R-TALEN left HA Tag 3) R-TALEN right myc Tag | ||
| isolation of plasmids | we isolated the two Plasmids 1) iGEM20? and 2) pET 32a+? out of E.coli with a QIAprep(R) Spin Miniprep Kit. The concentration was 1) 354 ng/ µl and 2) 43.5 ng/ µl dsDNA measured with a NanoPhotometer | ||
| 03.05.2016 | Janis | Assembly of TALEs, 2nd try | Cells could not be transformed, propably GGC of vectors did not work. Did the assembly again, this time over night. |
| PCR/Gels/Prep/Cloning | Primers arrived. Did PCR with N454-F/R from N454-HA and TRX_mod-F/R from PET32. PCR products cloned into PUC57-vector | ||
| 23.05.16 | Bianca | plasmidisolation | Plasmid isolation with QIAprep Spin Miniprep kit. The protocoll inside the kit was used without step 7. |
| Plasmids: TRXmodule1 (1), TRXmodule2 (2), iGEMN454-1 (3), iGEMN454-2 (4), 1-1, 1-2, 2-2, 3-1, 3-2, 5 FOK DS wos (without stop), 6 FOK RR wos, 7 Sc FOK wos | |||
| control digest | control digest with plasmids (5) - (9). Mastermix for (5)-(9): 3 µl EcoRI-HF, 3 µl BamHI-HF, 12 µl buffer CutSmart and 66 µl H2O, 14 µl of the Mastermix were added to 6 µl of each plasmid DNA | ||
| control digest with plasmids (1) - (4). Mastermix for (1)-(4): 5 µl BsaI 10 µl CutSmart, 55 µl H2O, 14 µl of the Mastermix were added to 6 µl of plasmid DNA | |||
| The digests ran for 1 h at 37 °C | |||
| agarose gel | after the digest 5 µl loadingbuffer was add to the control digest, 20 µl were loaded on a 1 % agarose gel for 30 min and at 140 V. 7 µl of a 1 kb DNA ladder were used. | ||
| 25.05.16 | Janis | sequencing | plasmids and primer were pipet together |
| 26.05.16 | Bianca | TNF reaction | 500 ng DNA, 1 µl Met, ad 50 µl H2O. Used DNA: iGEM 1-2 (A and B), MPAx SCL, 90 min at 30 °C |
| In-vitro-cleavage assay | TC(-): 12,5 µl DNA, 2 µl enzyme, 2 µl NEB3 buffer, 3,5 µl H2O (+) Ax7: 10 µl DNA, 2 µl enzyme, 2 µl NEB3 buffer, 6 µl H2O; 30 min at 37 °C | ||
| MiniElute PCR Purification Kit, 100 µl buffer PB, probes frozen with glycerin | |||
| 30.05.16 | Bianca | In-vitro-cleavage assay | 2 µl DNA ((+)AX7 und TC(-)), 3 µl NEB3 buffer, 5 µl BSA, 4 µl TNT(-)/Scl + 16 µl H2O or 13 µl TNT(+) + 7 µl H2O, ad 30 µl H2O |
| 1 h at 37 °C, 20 min at 65 °C, 10 min at 7000 xg centrifuget, 3 in at 14000 xg centrifuget, 20 µl supernatant + 5 µl LB | |||
| agarose gel | 1 % agarose gel, 50 ml liquide agarose + 5 µl RedSafe, 20 min at 140 V | ||
| Western blot preparing probes | at 37 °C, 30- (4 µl) and 30+ (13 µl) with 60 µl Laemmli, after 0, 30, 90 and 180 min. 10 min at 100 °C, store on ice | ||
| Repeat In-vitro-cleavage assay | SCL/TNT(+) with Ax7 | ||
| 25 µl TNT+, 4 µl buffer, 4 µl H2O, ad 40 µl H2O | |||
| 4 µl Scl, 4 µl buffer, 25 µl H2O, ad 40 µl H2O | |||
| 37 °C | |||
| 01.06.16 | Bianca | SDS gel | 15 µl probes for westernblot load on SDS gel (probes from 0, 30, 90 and 180 min + and -),40 min at 200 V |
| westernblot | 90 min at 120 mA | ||
| membran swing for 1 h in milkpouder and TBST, 1 x in TBST washed, 1 x 5 min in TBST washed, 1 x 10 min in TBST washed | |||
| membran with TBST and α-His (antibody) over night ( 15 µl antibody in TBST (100 µg/500 µl)) | |||
| 02.06.16 | Bianca, Janis | membran washed, second antibody | |
| 03.06.16 | Janis | transformation | chemically competent E.Coli cells were used, for Ax7L, Ax7R, scAx7L, 1-2; 2 µl DNA used and 50 µl competen cells; 20 min on ice, 45 sec at 42 °C, 1 h at 37 °C in 1 ml LB without antibitic. |
| cells with Ax7L, Ax7R, scAx7L plated on LB and ampicillin. cells with 1-2 on LB with chloramphenicol. Stored at 37 °C over night. | |||
| 07.06.16 | Wiebke, Theresa, Sabine | Interlab study | vector transformed into Top10 E.coli with the heatshock transfomation. 1 h at 37 °C |
| 27.06.16 | Bianca | Assembly of TALENs | new vector: p3H001; 4 rudiments with 1) Ax7L-DS, 2) Ax7R-RR, 3) Ax7L-scFOK, 4) Ax7R-scFOK |
| p3H001 77,0 ng/µl, 2 µl of C-terminal and 2 µl of N-terminal module, 5 µl H2O, used PCR programm (see protocol) | |||
| transformation | transformation into E.coli with electroporation. 200 µl LB and 30 min at 37 °C. Plated on LB chloramphenicol, stored at 37 °C over night. | ||
| 28.06.16 | Bianca | overnight cultures | 5 ml LB and 34 mg/ml chloramphenicol with 1 colonie from the plates. 3 culutres picked from each plate. Incubate at 37 °C over night. |
| 30.06.16 | Bianca, Kim | plasmid isolation | plasmid isolation with QIAprep Spin Miniprep kit. The protocoll inside the kit was used without step 7. Stored at -20 °C |
| overnight cultures | 3 colonies from each plate picked, overnight cultures with 5 ml LB, 34 mg/ml chloramphenicol. | ||
| 01.07.16 | Kim | control digest | control digest with the isolated plasmids from 30.06.16. 10 µl plasmid DNA, 2 µl 10x reaction buffer, 0,5 µl EcoRI, 0,5 µl PstI |
| 1 h at 37 °C and 10 min at 65 °C | |||
| agarose gel | loaded with 15 µl probes from control digest and 7 µl 1 kb plus DNA ladder | ||
| plasmid isolation | used the overnightcultures from 30.06.16; with QIA Prep Spin Miniprep Kit (without step 7) | ||
| 19.07.16 | Janis | retransformation | plasmids 1.1, 2.1, 3.1, 3.2, 4.1 in Top10 E.coli., incubation in LB with chloramphenicol (1.1: Ax7L-DS, 2.1: Ax7R-RR, 3.1: Ax7L-scFOK, 3.2: Ax7L-scFOK, 4.1: Ax7R-scFOK) |
| 20.07.16 | Kim | plasmid isolation | used the cultures from 19.07.16, with QIA Prep Spin Miniprep Kit (without step 7) |
| 21.07.16 | Janis | control digest | used plasmids from 20.06.16, with 4 µl plasmid DNA, 0,5 µl XbaI and 0,5 µl PstI, 2 µl buffer and 10 µl H2O. For 1 h at 37 °C and 10 min at 65 °C. |
| 28.07.16 | Sabine, Wiebke, Theresa | PCR | Run a PCR with 10 µl 5x HF Phusion Buffer, 5 µl dNTPs, 2,5 µl forward Primer, 2,5 µl reverse Primer, 1 µl template TCR5 H2, 0,5 µl Phusion polymerase, 28,5 µl H2O |
| 1. programm: 98 °C 2 min at the beginnig, denaturation at 98 °C for 10 sec, annealing at 60 °C for 10 sec, extension at 72 °C for 30 sec. steps repeated for 35 times.at the and 72 °C for 5 min. | |||
| 2. programm: same as the first programm, just the annealing temperature was 66 °C. | |||
| agarose gel | probes loaded on 1 % agarose gel, run for 120 V for 20 min, 1 kb plus DNA ladder. | ||
| transformation | 1.1, 2.1, 3.1, 3.2 and 4.1 in BL21DE3 E.coli with electroporation. 1 h at 37 °C , plated on LB with chloramphenicol, inkubated at 37 °C | ||
| puc57 in Top10 with heatshock at 42 °C for 40 sec, 200 µl LB added, 1 h at 37 °C. Plated on LB | |||
| gel elution | cut the fragment out of the gel. add 300 µl QG buffer, 10 min at 50 °C, add 100 µl Isopropanol, transfered to column, 1 min at max. rpm, add 400 µl QG buffer, 1 min at max. rpm, add 700 µl PE buffer, | ||
| 1 min at 1500 rpm, centrifuge to dry the column, 1 min at max rpm, add 15 µl EB buffer or H2O, incubate for one minute, centrifuge for 1 min at max rpm. | |||
| Cut and ligation | 2 µl cut smart buffer, 2 µl ATP, 2 µl puc57, 1 µl SmaI, 1 µl T4 Ligase, 15 µl Elution, 2 µl H2O, for 2 h at 30 °C | ||
| PCR | repeat the PCR, but used 1 min elonation time at 72 °C | ||
| agarose gel | load gel with PCR probes, run at 120 V for 20 min | ||
| 01.08.16 | Wiebke, Sabine | Plasmid isolation | Plasmid isolation from overnightcultures with iGem Nterm and GFP puc57 (1, 2, 3, 4) |
| Used QIA Prep Spin Miniprep Kit without step 7. 4 ml Overnightculture were used. | |||
| control digest | with the isolated plasmids. used 0,5 µl BsaI, 7 µl DNA, 2 µl CutSmart buffer, ad 20 µl (10.5 µl H2O) | ||
| 1 h at 37 °C, 10 min at 65 °C. | |||
| agarose gel | 1 % agarose gel, used 7 µl 1 kb DNA ladder, 20 µl from Controll digest with 6 µl 5x LD buffer (20 µl loaded on agarose gel) | ||
| Gel at 120 V for 25 min | |||
| 02.08.16 | Wiebke, Sabine, Louise | plasmid isolation | Repeat the plasmid isolation from 01.08.16 |
| control digest | with the isolated plasmids. used 1 µl BsaI, 26 µl DNA, 3 µl CutSmart buffer | ||
| agarose gel | 1 % agarose gel, used 7 µl 1 kb DNA ladder, 20 µl from Controll digest with 6 µl 5x LD buffer (20 µl loaded on agarose gel) | ||
| 03.08.16 | Janis | ||
| cloning | GFP - NTH3 Fragment into puc57: 5 µl DNA, 1 µl SmaI, 1 µl T4 Ligase, 1 µl Puc57, 1 µl ATP, 1 µl tango buffer; 1 h at room temperatur (RT) | ||
| transformation | puc57 with GFP - NTH3 in E.coli (Top10) | ||
| transformation | transformation into E.coli (BL21DE3) with electroporation. 200 µl LB and 30 min at 37 °C. Plated on LB chloramphenicol, stored at 37 °C over night. | ||
| Used the DNA 1.1, 2.1, 3.1, 3.2 and 4.1 | |||
| 10.08.16 | Kim | overnightcultures | used 4 colonies from 03.08. (puc57 with GFP-NTH3 in Top10), 5 ml LB, 5 µl carbenicillin. |
| 11.08.16 | Kim | plasmid isolation | plasmid isolation with the overnightcultures from 10.08.16, used 4 ml overnightculture and QIA Prep Spin Miniprep Kit without step 7. |
| 12.08.16 | Kim | control digest | plasmids from 11.08.16; 5 µl DNA, 2 µl CutSmart buffer, 0.5 BsaI, 12.5 µl H2O; 1 h at 37 °C, 10 min at 65 °C |
| agarose gel | 1 % agarose gel loaded with 20 µl probe (20 µl control digest and 4 µl 6x loading buffer), 7 µl 1 kb DNA ladder; 27 min at 100 V | ||
| 16.08.16 | Theresa, Lousie, Severin | PCR | like the PCR from 28.07.16 |
| agarose gel | 1 % agarose gel loaded with 60 µl probe (50 µl PCR probe and 12,5 5x loading buffer), 8 µl DNA ladder; 20 min at 120 V | ||
| gel extraction | gel extraction with MiniElute Extraction Kit | ||
| cloning | like the cloning from 03.08.16, but CurSmart buffer was used | ||
| transformation | 10 µl cloning stuff, 50 µl competent E.coli cells; add 200 µl LB for 30 min | ||
| 10 µl puc57, 50 µl competent E.coli cells; add 200 µl LB for 30 min | |||
| 17.08.16 | Janis | overnightcultures | 5 colonies (1-5) picked from transformation (16.08.16), in 5 ml LB with 5 µl Carbenicellin |
| 18.08.16 | Janis | plasmid isolation | plasmid isolation (overnightcultures used from 17.08.16) with QIA Prep Spin Miniprep Kit without step 7. |
| control digest | control digest with the isolated plasmids (18.06.16), same control digest like the digest from 12.08.16 (5 µl DNA, 2 µl CutSmart buffer, 0.5 BsaI, 12.5 µl H2O; 1 h at 37 °C, 10 min at 65 °C) | ||
| assembly | assembly oft GFP-NTH3 into iGem vector; 2 µl iGem vector (50 ng/µl), 1 µl hexa-repeat LA, 1 µl hexa-repeat AB, 1 µl hexa-repeat BR (each 50 ng/µl), 2 µl N-terminal module (100 ng/µl), 2 µl C-terminal module (100 ng/µl), 2 µl NEB buffer, | ||
| 1 µl enzym Mix (2 µl ATP, 2 µl NEB CutSmart, 1 µl T4-DNA Ligase, 1 µl BsaI) and 7 µl H2O | |||
| assembly program: 10 min at 40 °C, 10 min at 16 °C (3 cycles), 20 min at 50 °C, 20 min at 80 °C (Final) | |||
| transformation | 15 µl assembly, 50 µl competent cells, transformation with electroporation, 200 µl LB, 1 h at 37 °C, plated on LB with chloramphenicol | ||
| 22.08.16 | Kim | overnightcultures | picked 6 colonies from 18.08.16, with LB and Chloramphenicol |
| 23.08.16 | Kim | plasmid isolation | plasmid isolation (used 4 ml overnightcultures from 22.08.16) with QIA Prep Spin Miniprep Kit without step 7 |
| control digest | with plasmids (23.08.16), used 7 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 10 µl H2O | ||
| 1 h at 37 °C, 10 min at 65 °C | |||
| agarose gel | 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V | ||
| 24.08.16 | Janis, Kim | transformation | plasmids 1.1, 2.1, 3.1, 3.2, 4.1 in BL21 E.coli., incubation on LB with Chloramphenicol at 37 °C |
| control digest | repeat control digest from 23.08.16 with 10 µl DNA (2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 7 µl H2O); 1 h at 37 °C, 10 min at 65 °C | ||
| agarose gel | 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V | ||
| overnightculures | picked 4 colonies from 18.08.16, with LB and Chloramphenicol | ||
| 25.08.16 | Janis, Kim | plasmid isolation | plasmid isolation (overnightcultures used from 24.08.16 7-10(GFP-NTH3 in iGem vector)) with QIA Prep Spin Miniprep Kit without step 7. |
| plasmid isolation (overnightcultures interlab study + control 1, + control 2, - control 1, - control 2) | |||
| transformation | retransformation plasmids 1.1, 2.1, 3.1, 3.2 and 4.1 in Top 10 E.coli, 50 µl competent cells, 20 min on ice, 45 sec at 42 °C, 1 h at 37 °C in 200 µl LB, plated on LB with chloramphenicol | ||
| transformation plasmids for interlab study in Top10 E.coli, 50 µl competent cells, 20 min on ice, 45 sec at 42 °C, 1h at 37 °C in 200 µl LB, plated on LB | |||
| 26.08.16 | Kim | control digest | with plasmids (25.08.16), used 11 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 6 µl H2O - different XbaI and PstI stocks used, also repeat control digest with plasmids 1-6 |
| 1 h at 37 °C, 10 min at 65 °C | |||
| agarose gel | 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V | ||
| 29.08.16 | Bianca | Interlab study, transformation | DNA from Göttingen in 10 µl H2O; 50 µl competent cells (Top10 E.coli), 2 µl DNA, 20 min on ice, 45 sec at 42 °C, 1 ml LB 1 h at 37 °C, plated on LB with chloramphenicol, incubate at 37 °C |
| overnightcultures | 3 colonies each plate (from 25.08.16, Top with 1.1, 2.1, 3.1, 4.1), with 5 ml LB and Chloramphenicol, incubate at 37 °C | ||
| 3 colonies each plate (from 24.08.16, BL21 with 1.1, 2.1, 3.1, 3.2, 4.1) with 5 ml LB and Chloramphenicol, incubate at 37 °C | |||
| 30.08.16 | Bianca, Theresa | expression | 2.5 ml from each overnightculture (from 29.08.16, BL21 with 1.1, 2.1, 3.1, 3.2, 4.1) in 10 ml LB |
| Incubation, optical density after 60 min: 1.1 - 0.136, 2.1 - 0.122, 3.1 - 0.181, 3.2 - 0.153, 4.1 - 0.134 | |||
| optical density after 115 min: 1.1 - 0.489, 2.1 - 0.494, 3.1 - 0.596, 3.2 - 0.520, 4.1 - 0.420 | |||
| optical density after 150 min: 1.1 - 0.728, 2.1 - 0.540, 3.1 - 0.921, 3.2 - 0.792, 4.1 - 0.660 | |||
| 1 ml, centrifuged, pellet stored at -20 °C (pre induction) | |||
| add 238 µl IPTG, incubate for 1 h and 40 min at 37 °C, 1 ml centrifuged, pellet stored at -20 °C (after induction) | |||
| rest centrifuged, into liquid nitrogen and stored at -80 °C | |||
| plasmid isolation | plasmid isolation (overnightcultures re-transformation from 29.08.16) with QIA Prep Spin Miniprep Kit without step 7 | ||
| overnightcultures | 100 µl overnightculture (from 26.08.16 interlab study) with 8 ml LB and chloramphenicol, incubate at 37 °C | ||
| 31.8.16 | Bianca, Theresa, Janis | assembly | with 1 µl GFP-NTH3, 1 µl vector (50 ng/µl), 1 µl HexaI (Repeats 1-6), 1 µl HexaII (Repeats 7-11,5), 1 µl T4-Ligase, 1 µl BsaI, 2 µl 10mM ATP, 2 µl CutSmart buffer, ad 20 µl H2O |
| two different vectors were used: iGem vector (with c-terminus c-63) and psaE6 (with c-terminus c-17) and 4 different Hexa combinations each vector | |||
| A: iGem vector and Hexa 42 + HDHx | |||
| B: iGem vector and Hexa 42 + 2xNG | |||
| C: iGem vector and Hexa 42 + 2xNN | |||
| D: iGem vector and pJet Hexa3 rep | |||
| E: psaE6 and Hexa 42 + HDHx | |||
| F: psaE6 and Hexa 42 + 2xNG | |||
| G: psaE6 and Hexa 42 + 2xNN | |||
| H: psaE6 and pJet Hexa3 rep | |||
| interlab study | culture and measurment | ||
| 01.09.16 | Janis, Theresa | transformation | 15 µl assambly product, 50 µl competent cells (E.coli, Top10), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 1 h at 37 °C, plated on LB with Chloramphenicol |
| SDS gel | prepared 4 gels | ||
| control digest | with plasmids (25.08.16, re-transformation), used 11 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 6 µl H2O | ||
| 1 h at 37 °C, 10 min at 65 °C | |||
| 05.09.16 | Theresa, Kim | SDS gel | prepared 8 gels |
| separating gel (10 %): 27.25 ml H2O, 33.25 Acrylamid (30 %), 25 ml Tris (1.5 M; pH 8.8), 1 ml APS (10 %), 1 ml SDS (10 %), 40 µl TEMED | |||
| stacking gel: 34 ml H2O, 8.5 ml Acrylamid (30 %), 6.25 ml Tris (0.5 M; pH6.8), 0.5 ml APS (10 %), 0.5 ml SDS (10 %), 50 µl TEMED | |||
| first separating gel, than stacking gel; ~1 ml Isopropanol on separating gel, when it is in the chamber (remove it before filling stacking gel in the chamber) | |||
| 1. H2O, 2. Tris, 3. SDS, 4. Acrylamid, 5. APS, 6. TEMED; APS and TEMED everytime at last! After adding them be fast | |||
| overnightcultures | with colonies from tranformation 1.09.16, 4 colonies of the plates A-D, there was nothing on the plates E-H; 5 ml LB, 5 µl Chloramphenicol and the picked colonie | ||
| 06.09.16 | Theresa, Kim | plasmid isolation | plasmid isolation (overnightcultures from 05.09.16, A1-3, B1-3, C1-3, D1-3) with QIA Prep Spin Miniprep Kit without step 7 |
| transformation | used plasmids from assembly F, G and H (31.08.16), 10 µl assambly product, 50 µl competent cells (E.coli, Top10), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 30 min at 37 °C, plated on LB with Chloramphenicol | ||
| 07.08.16 | Theresa, Bianca, Kim | control digest | with plasmids (25.08.16), used 4 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 13 µl H2O |
| 1 h at 37 °C, 10 min at 65 °C | |||
| agarose gel | 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V | ||
| SDS gel | prepared probes: took a little bit of the pellets (from the expression on 30.08.16), also the pre-induction and the after-induction pellets; 80 µl Leammli, resuspent, 10 min at 100 °C | ||
| load gel with 15 µl of the prepared probes, 5 µl Page Ruler best; run for 42 min; gel in coomassi staining solution | |||
| transformation | 15 µl plasmid DNA (from plasmid isolation 06.09.16, plasmids A3, A4, B1, B2, C2, D1, D2) 50 µl competent cells (E.coli, BL21), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 35 min at 37 °C, 100 µl plated on LB with Chloramphenicol | ||
| 08.09.16 | Theresa, Kim | SDS gel | gel 3 h in H2O and 4 h in destaining solution, after that over night in PBS buffer |
| transformation | repeat transformation from 07.08.16, nothing changed | ||
| 09.09.16 | Kim | overnightcultures | used plates from transformation (07.09.16), each plate (A3, A4, B1, B2, D2) one colonie picked, 5 ml LB and 5 µl Chloramphenicol (34 mg/ml) |
| 12.09.16 | Kim | overnightcultures | used plates from transformation (07.09.16), each plate (A3, A4, B1, B2, C2, D2) one colonie picked, 5 ml LB and 5 µl Chloramphenicol (34 mg/ml) |
| 13.09.16 | Theresa, Sabine, Wiebke | Expression | Used the overnightcultures from 12.09.16, 100 ml LB and different amounts of overnightcultures, depents on the optical density. Used 1.17 ml A, 1.8 ml B, 3.22 ml C, 2.4 ml D - Start optical density was 0.05 |
| Incubation; optical density after 1 h: A - 0.063, B - 0.11, C - 0.201, D - 0.115 | |||
| Optical density after 2.5 h: A - 0.452, B - 0.59, C - 0.93, D - 0.756 | |||
| Optical density after 2.75 h: A - 0.5 | |||
| Optical density after 3 h: A - 0.59 | |||
| 1 ml, centrifuged, pellet stored at -20 °C (pre induction) | |||
| add 200 mM IPTG (1 ml), incubate for 1 h and 30 min at 37 °C, 1 ml centrifuged, pellet stored at -20 °C (after induction) | |||
| rest centrifuged 10 min, into liquid nitrogen and stored at -80 °C | |||
| 14.09.16 | Sabine, Wiebke, Louise, Theresa, Kim, Severin | Purification of tales | Resuspensionof sample 1.1.1in 2ml TE-Buffer and 20 μl PMSF |
| Ultrasonic treatment on ice: program9, 30%energy for 8 min (30sec on/30 sec off) | |||
| Centrifuged at 4°C, 20.000gfor 15 min | |||
| Supernatant transferred to column(Gravity flow Strep Tactin PepharoseColumn, Iba), purification as | |||
| stated by iba protocol | |||
| Flow through was stored as aliquots of 500µl labelled ‘E 1 – 6’ and ‘W 1 – 5’. Flow through of first centrifugation was stored and labelled as ‘supernatant’. Storage at -20°C. | |||
| 15.09.16 | Theresa, Wiebke, Kim, Sabine, Severin | SDS-Gels with samples derived from TALe 1.1.1 (14.09.2016) | 50µl of each sample mixed with 5,88µl TCA (final concentration 10%). Incubated for 10 min on ice. Centrifuged at 4°C, 20.000g for 10 min. Mixed with 500µl of 80% acetone and centrifuged at 4°C, 20.000g for 10 min. |
| Incubated for 30 min at 37°C. Mixed with 40µl Laemmli and incubated for 10min at 80°C. Gels ran for 1 h and 20 min at 200V in TANK buffer. | |||
| Coomassie-Staining | Coomassie-gels were set to stain over night over night. | ||
| Western Blot | Other gels were Blotted semi-dry at 70V for 50 min. Poncau-staining was done for 10min. Incubated for 1 h in PBST with 3% milkpowder, Incubated over night with 1st antibody | ||
| (StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science) | |||
| 16.09.16 | Sabine, Kim, Wiebke | Continuation of Western | 1st Antibody removed by washing in PBST, Incubated in PBST with 2nd antibody (Ecl anti-mouse IgG horseradish peroxidase) for 3 h |
| Stained with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22.2 mg/ 500 µl DMSO), 15µl p-Coumaric acid (7.4 mg/ 500 µl DMSO) and 10µl 30% H2O2 for 1 min up to 30 min | |||
| Continuation of Coomassie-Staining | Destained for 1,5 h and incubated in water for 1h | ||
| 20.09.16 | Sabine, Wiebke, Kim, Theresa, Bianca, Louise | Purification of tales | Resuspension of sample 1.1.1, B2.1 and C2.1 in 2 ml TE-Buffer and 20 µl PMSF, Ultrasonic treatment on ice: program 9, 30% energy for 8 min (30 sec on/30 sec off), Centrifuged at 4°C, 20.000g for 15 min |
| Supernatant transferred to column (Gravity flow Strep Tactin Pepharose Colum, Iba), purification as stated by Iba protocol | |||
| Flow through was labelled as ‘E 1 – 6’ and ‘W 1 – 5’ and stored at -20°C | |||
| Others | Fluorecence of GFP-TAL fusion proteins in BL21 strains 1.1.1, B1 and C2 and in corresponding raw extracts was documented | ||
| Expression | Overnight cultures in 200ml LB of BL21 strains B2 and C2 (12.09.2016) inoculated and incubated at 37°C | ||
| Overnightcultures | Overnight cultures in 5ml LB and master dish of 2.1.1, 3.1.1, 3.2.1 and 4.1.1 (12.09.2016) inoculated and incubated at 37°C | ||
| 21.09.16 | Bianca | TEV digestion | According to ProTEV plus protocol (Promega) |
| 2µl Buffer 0,4µl 100mM DTT 36,8µl Protein (E6,5) as collected on 14.09.2016 0,8µl TEV |
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| Incubated at 30°C. Samples of 9,5µl were taken after 0, 1, 2, 4 and 6h and stored at -20°C | |||
| Proteinexpression | Overnight cultures of BL21 strains B2 and C2 (20.09.2016) were used to inoculate 5x 200ml LB+CA each and grown at 37°C to an OD600 of approx. 0,500. | ||
| 1ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20°C. | |||
| Proteinexpression was induced with 400 µl IPTG, final concentration: 2mM | |||
| After 1,5h 1ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20°C | |||
| and the remaining cells were harvested and stored at -80°C | |||
| TCA precipitation | Samples derived from 1.1.1, B2.1 and C2.1 (20.09.2016) were mixed with 3µl NaDOC and incubated for 10min on ice | ||
| 3µl TCA were added and samples were incubated for 60min on ice, Centrifuged at 4°C, 6,500RPM for 20 min, Pellets were resuspended in 1ml 80% acetone, | |||
| Centrifuged at 4°C, 6,500RPM for 10 min, Supernatant was discarded; samples were dried at 37°C, 30µl SDS running-buffer + Lämmli was added, incubated at 80°C for 10min, Samples were stored at -20°C | |||
| other | Overnight culture of 1.1.1 was transferred into 30ml fresh LB+CA and incubated over night at 37°C | ||
| 22.09.16 | Bianca, Janis | Proteinexpression | Overnight culture of 1.1.1 (21.09.2016) was used to inoculate 5x 200ml LB+CA each and grown at 37°C to an OD600 of approx. 0,600. 1ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20°C. |
| Proteinexpression was induced with 400 µl IPTG, final concentration: 2mM | |||
| After 1,5h 1ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20°C and the remaining cells were harvested and stored at -80°C | |||
| SDS-GELS | Gels ran for 40 min at 200V in TANK buffer | ||
| Western Blot | semi-dry blot at 6,9V for 45 min | ||
| Poncau-staining was done for 10min | |||
| Incubated for 1 h in PBST with 3% milkpowder | |||
| Incubated at 4°C over night with 1st antibody (StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science | |||
| Plasmidisolation, With Quigen Mini Prep Spin Column Kit according to protocol. | |||
| Masterdish | Petridish was damaged, redone with recent BL21 strains: A3.1, A3.2, A3.3, A4.1, A4.2, A4.3, B1.1, B1.2, B1.3, B2.1, C2.1, C2.2, C2.3, D1.1 | ||
| 23.09.16 | Bianca, Janis, Kim | Control digest | Plasmids isolated on 22.09.2016 were digested with PstI and XbaI (Set1) as well as PstI, XbaI and BsaI (Set2) |
| Continuation of Western Blot | 1stAntibody removed by washing in PBS, Inbubation with 2nd, Incubated in PBST with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for 2h, 2nd Antibody removed by washing in PBS, | ||
| Stained with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 for 1 min up to 30 min | |||
| 26.09.16 | Kim, Bianca | SDS-Gel | Used premade 10% Gel from Biorad, TEV-digestion (21.09.2016) samples were mixed with 10µl Lämmli each, E6 1.1.1, B, C (20.09.2016) samples were mixed with 30µl Lämmli each, Incubation at 100°C for 10min |
| Gels ran for 45min at 150V in TANK buffer. | |||
| Western Blot | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) Blotted at 6V for 40min in fastBlot Trans Blot Turbo | ||
| Poncau-staining was done for 10min | |||
| Incubated for 1h in blocking solution (1xPBST with 1xRotiBlock, Roth), washed with PBST | |||
| Incubated at 4°C over night with 1st antibody | |||
| Coomassie staining | Coomassie-gels were set to stain over night | ||
| Overnight cultures | 3.1.1 and 4.1.1 were inoculated in 25ml LB+CA and grown overnight at 37°C | ||
| 27.09.16 | Bianca, Kim, Wiebke | TEV digestion | According to ProTEV plus protocol (Promega) |
| 2µl Buffer, 0,4µl DTT, 36,8µl Sample (1.1.1 E6 as collected on 20.09.2016), 0,8µl TEV | |||
| Incubated at 30°C. Samples of 9,5µl were taken after 0, 1, 2, 4 and 6h and stores at -20°C | |||
| Continuation of Coomassie-Staining | After incubation with destaining solution no bands, not even marker, were visible. Gel discarded. | ||
| Continuation of Western Blot | 1stAntibody removed by washing in PBS | ||
| Incubated in PBST with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for 1h | |||
| 2nd Antibody removed by washing in PBS | |||
| Stained with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| Proteinexpression | Overnight cultures of BL21 3.1.1 and BL21 4.1.1 (26.09.2016) was used to inoculate 50ml LB+CA each and grown at 37°C to an OD600 of approx. 0,600. | ||
| 1ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20°C. | |||
| Proteinexpression was induced with 100 µl IPTG, final concentration: 2mM | |||
| After 1,5h 1ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20°C and the remaining cells were harvested and stored at -80°C | |||
| 28.09.16 | Kim, Bianca | Purification of tales | Resuspension of samples 1.1.1 (22.09.2016), B and C (21.09.2016) as well as 3.1.1 and 4.1.1 (27.09.2016) in 2 ml TE-Buffer and 20 µl PMSF, Ultrasonic treatment on ice: program 9, 30% energy for 8 min (30 sec on/30 sec off) |
| Centrifuged at 4°C, 20.000g for 15 min | |||
| Supernatant transferred to column (Gravity flow Strep Tactin Pepharose Colum, Iba), purification as stated by Iba protocol | |||
| 29.09.16 | Janis, Bianca, Kim | SDS-Gel | Proben: Raw extract B from 21.09., E6 B from 20.09., E6 B from 28.09. |
| Raw extract C from 21.09., E6 C from 20.09., E6 C from 28.09. | |||
| Raw extract 1.1.1 from 22.09., E6 1.1.1 from 20.09., E6 1.1.1 from 28.09. | |||
| Raw extract 3.1.1, E6 3.1.1 from 28.09. | |||
| raw extract 4.1.1, E6 4.1.1 from 28.09 | |||
| TEV 0 from 27.09., TEV 1 from 27.09., TEV 2 from 27.09., TEV 4 from 27.09. | |||
| Coomassie-Staining | Incubation overnight at RT | ||
| Western Blot | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)), Blotted at 6V for 40min in fastBlot Trans Blot Turbo | ||
| Poncau-staining was done for 10min | |||
| Incubated for 1h in PBST with 3% milk powder, washed with PBST | |||
| 30.09.16 | Kim | Continuation of Coomassie-Staining | Destained Gels |
| Continuation of Western Blot | Washed membrane with PBS | ||
| Incubated with 1st antibody (StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science) for 1h | |||
| 1stAntibody removed by washing in PBS | |||
| Inbubation with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for one hour | |||
| 2nd Antibody removed by washing in PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| 04.10.16 | Kim, Severin, Wiebke | Colonie-PCR | Single Colonies of B, C and 1.1.1 were suspended in 10µl H2O and incubated at 95°C for 5min. |
| 1µl sample and 49µl mastermix were prepared and amplified as follows: | |||
| 50µl Buffer 5xG | |||
| 5µl dNTPs | |||
| 10µl Primer 1 (1534) | |||
| 10µl Primer 2 (1716) | |||
| 7.5µl DMSO | |||
| 135µl H2O | |||
| 25µl MgCl2 | |||
| Program: | |||
| 35x (30s 98°C, 30 s 59 °C, 30 s 72 °C) and 5 min 72 °C | |||
| agarose gel | 1 % agarose Gel, Gels ran for 27min at 100V | ||
| Proteinexpression | 1ml LB+CA was inoculated with strain C as a preculture and later distributed into 10x 10ml LB. | ||
| Cultures were grown to an OD600 of approx. 0,5 as well as 0.847 | |||
| Proteinexpression was induced at 30°C and 37°C with 2mM IPTG for as long as 3h and over night. | |||
| 05.10.16 | Kim, Severin, Wiebke | Liquid cultures were harvested and split into two separate reaction tubes for Dotplot and Immunostain | |
| 06.10.16 | Kim, Wiebke | TEV digestion | Using samples according to ProTEV plus protocol (Promega) |
| 5µl Buffer | |||
| 1µl 100mM DTT | |||
| 40µl Sample 1.1.1 (28.09.2016) and C (20.09.2016, 20µl E5 and 20µl E6) | |||
| 2µl TEV | |||
| 52µl H2O | |||
| Incubated at RT. Samples of 20 µl were taken after 0, 1, 2, 4 and 6h and stored at -20°C | |||
| SDS-Gel | 20µl of TEV digestion sample was mixed with 20µl Lämmli and incubated at 80°C for 10min, Gels (10%) ran for 50min at 150V in TANK buffer. | ||
| Western Blot | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) | ||
| Blotted at 25V for 40min in fastBlot Trans Blot Turbo | |||
| Poncau-staining was done for 10min | |||
| Washed with H2O | |||
| Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth) | |||
| Washed with PBS | |||
| Incubated at 4°C over night with 1st antibody | |||
| 07.10.16 | Wiebke, Severin, Kim | Continue the Immunostain. | Wash the Membran with PBS |
| Incubate at RT with the second Antibody for one hour | |||
| Wash with PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8.5), 30µl Luminol (22.2ng/500µl DMSO), 15µl p-Coumaric acid (7.4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| Spotting the Oligonucleotides on a Chip | iGEM.npw as layout | ||
| Solutions on Lafontain plates, 10 µl Oligonucleotides (100 µM) + 10 µl array it Micro spotting (2x) | |||
| spotting | |||
| Aftertreatment: | |||
| 1) UV-Crosslink for 3 minutes | |||
| 2) 24 hours in a dark room at room temperature (RT) | |||
| 3) reducing: 2 min 0.2 % SDS, 2x 1 min H2O, 5 min NaBH4, 1 min icecold H2O, 1 min 0.2 % SDS, 2x 1 min H2O | |||
| dry | |||
| 4) Blocking: 45 min Blocking reagent, 42 °C 5 min H2O | |||
| dry | |||
| Overnightcultures | Used glycerin stock from Origami cells, incubate in 20 ml LB medium at RT over the weekend | ||
| Used colonies from 1.1.1 and 2.1.1 from masterdish, 50 ml LB medium and 50 µl Chloramphenicol. Incubate over the weekend at RT | |||
| 10.10.16 | Kim, Wiebke, Severin | Purification | Samples of 1.1.1 (20.09.2016) and 2.1.1 (30.08.2016) were resuspended in 5ml TE-Buffer with 50µl PMSF. Proteins were purifies according to Iba protocol |
| One Column of 2.1.1 was incubated over night with 50mM DTT. (at the 5th washing step, use 2 column bead volume (0.4 ml) and add 50 mM DTT. Incubate over night) | |||
| Proteinexpression | Cultures of 1.1.1 and 2.1.1 were inoculated into 5x 200ml LB+CA | ||
| Cultures were grown to an OD600 of approx. 0,5-0,6 | |||
| Proteinexpression was induced at 30°C and 37°C with 2mM IPTG for as long as 1,5h. | |||
| Afterwards cells were harvested and stored at -80°C | |||
| Prepation of electro competent cells | Take 1 ml or more from the overnight culture from origami cells into 50 ml LB medium. Grow the cells until they reaches a optical density at 600 nm of 0.5 – 0.6. Incubate the bacteria 20 min on ice. | ||
| Centrifuge 10 min at 4 °C and discard the supernatant. Add 20 ml cold H2O and resuspend the pellet. Centrifuge again 10 min at 4 °C. | |||
| Add 20 ml cold H2O and centrifuge for 10 min at 4 °C. Discard the supernatant and add 20 ml cold H2O. Centrifuge for 10 min at 4 °C. Discard the supernatant and add 10 ml cold glycerin (10 %). | |||
| Resuspend the pellet and centrifuge at 4 °C for 10 min. discard the supernatant and resuspend the pellet in 2 ml 10 % cold glycerin | |||
| Take 100 µl aliquots into 1.5 µl reaction tubes. Froze them in liquid nitrogen and store at - 80 °C or use them directly for transformation. | |||
| transformation | electropotation of competent origami cells with 1 µl plasmid DNA (from 1.1.1 and 2.1.1) | ||
| incubate for 30 min in 500 µl LB at 37 °C. Plate 100 µl on LB with Chloramphenicol. incubate at 37 °C over night | |||
| 11.10.16 | Wiebke, Louise, Kim | Continued Purification | More samples of 1.1.1 (20.09.2016) and 2.1.1 (30.08.2016) were resuspended in 5ml TE-Buffer with 50µl PMSF. Proteins were purifies according to Iba protocol |
| One Column of 1.1.1 and 2.1.1 were incubated over night with 50mM DTT. | |||
| the incubate column from 10.10.16 were Eluated | |||
| Stability test of circularised TALes | Purified protein samples of 2.1.1 were set to incubate with DTT (circularised) or without (control) for 0, 6, 24, 32, 48 h at 4°C, RT and 37°C. | ||
| In addition, one set of samples was frozen at –20°C and thawed at RT for 30min each for four continuous times. | |||
| The linearized TAL 3HD100 was used as an additional control. After each time a fraction of 10µl was stored at -20°C for analysis. | |||
| SDS-Gel | 10µl of sample were mixed with 10µl Lämmli and incubated at 80°C for 10min. | ||
| 10µl of the sample/Lämmli mixture were put onto the Gels (10%) which ran for 50min at 150V in TANK buffer. | |||
| western blot | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) | ||
| Blotted at 25V for 40min in fastBlot Trans Blot Turbo | |||
| Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth) | |||
| Washed with PBS | |||
| Incubated at 4°C over night with 1st antibody | |||
| Overnightculture | Used colonies from 1.1.1 and 2.1.1 fromtransformation with origami, 20 ml LB medium and 20 µl Chloramphenicol. Incubate at 37 °C over night | ||
| 12.10.16 | Wiebke, Louise, Sabine, Kim | Proteinexpression | Cultures of 1.1.1 and 2.1.1 were inoculated into 2x 125ml LB+CA |
| Cultures were grown to an OD600 of approx. 0,5-0,6 | |||
| Proteinexpression was induced at 30°C and 37°C with 2mM IPTG for as long as 2h. | |||
| Afterwards cells were harvested and stored at -80°C | |||
| TEV digestion | Using samples 1.1.1 +/- DTT (11.10.2016) and 2.1.1 +/- DTT (11.10.2016) according to ProTEV plus protocol (Promega) | ||
| Samples were taken after 0, 1, 2, 4 and 6h and stored at -20°C | |||
| Continue Immunostain | wash membran with PBS | ||
| Incubate second antibody for 1 hour | |||
| wash with PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8.5), 30µl Luminol (22.2ng/500µl DMSO), 15µl p-Coumaric acid (7.4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| 13.10.16 | Wiebke, Kim, Sabine, Louise | SDS-Gel | TEV digested samples (12.10.2016) were mixed 1:1 with Lämmli and incubated at 80°C for 10min. |
| Gels (10%) ran for 50min at 150V in TANK buffer. | |||
| Stability test of circularised TALes | Purified protein samples (13.10.2016), 1.1.1 (E5 + E6) and 2.1.1 (E5 + E6), were set to incubate for 0, 15min, 5h and 20h at 4°C, RT and 37°C. | ||
| In addition, one set of samples was frozen at –20°C and thawed at RT for 30min each for four continuous times. Each time a fraction of 10µl was stored at -20°C for analysis. | |||
| Western Blot and Immunostain | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) | ||
| Blotted at 25V for 40min in fastBlot Trans Blot Turbo | |||
| Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth) | |||
| Washed with PBS | |||
| Incubated for 3h at RT with 1st antibody (2 times Flag antibody, to times Strep antibody at TEV digest Western blot) | |||
| 14.10.16 | Wiebke, Kim, Sabine | Continue Immunostain | 1stAntibody removed by washing in PBS |
| Inbubation with 2nd antibody | |||
| 2nd Antibody removed by washing in PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| Stability test of circularised TALes | Purified protein samples (13/14.10.2016) of Origami with DTT were set to incubate for 6 h, 48 h ... at 4°C, RT and 37°C. | ||
| In addition, one set of samples was frozen at –20°C and thawed at RT for 30min each for four continuous times. Each time a fraction of 10µl was stored at -20°C for analysis. | |||
| Using samples E1 – E6 from 13.10.16 according to ProTEV plus protocol (Promega) | |||
| Samples were taken after 0, 1, 2, 4 and 6h and stored at -20°C | |||
| 16.10.16 | Wiebke | Stability Test | Probe at 4 pm (68 h for probes without DTT and 54 h for the probes with DTT) |
| 17.10.16 | Wiebke, Louise, Sabine, Theresa, Kim | SDS gels | Used 8 SDS Gels for the probes from the two stability tests with and without DTT. One gel was used for the TEV digest. |
| Gels run for 50 min at 150 V. | |||
| Western blot | Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) | ||
| Blotted at 25V for 40min in fastBlot Trans Blot Turbo | |||
| Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth) | |||
| Washed with PBS | |||
| Immunostain | Incubated Membrane 1-6 for 2 h at RT with 1st antibody and membrane 7-9 over Night at 4 degree | ||
| 1stAntibody removed by washing in PBS | |||
| Inbubation with 2nd antibody for 1 h | |||
| 2nd Antibody removed by washing in PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 | |||
| 18.10.16 | Kim | Continue Immunostain from 17.10.16 | 1stAntibody removed by washing in PBS |
| Inbubation with 2nd antibody for 1 h | |||
| 2nd Antibody removed by washing in PBS | |||
| Stained for 1min with 6ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2ng/500µl DMSO), 15µl p-Coumaric acid (7,4ng/500µl DMSO) and 10µl 30% H2O2 |
Protocols
