Revision as of 05:48, 19 October 2016

Notebook

Protocols

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
			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

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Difference between revisions of "Team:Hannover/Notebook"

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