Team:PKU Beijing/Notebook/AND Gate 1/Input/LacI TetR 2

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Notebook > AND Gate 1 > Input > Molecular cloning: RBS-lacI/tetR + terminator

Molecular cloning: RBS-lacI/tetR + terminator

PKU Shuke Wu LacI TetR 2.JPG

Resource:
RBS-LacI: myself: 2×B0034-C0012, renamed as L1, L2
RBS-tetR: myself: 2×B0034-C0040, renamed as t2, t3
Terminator: Haoqian Zhang & Guosheng Zhang: B0015

2009.7.10

Plasmid mini prep:
6 RBS-tetR: RBS1-tetR1; RBS2-tetR3; RBS3-tetR2, 3 (t2, t3); RBS4-tetR2; RBS5-tetR2;
6 RBS-lacI: RBS1-lacI1; RBS2-lacI1; RBS3-lacI1, 2 (L1, L2); RBS4-lacI3; RBS5-lacI2;

Double digest:
L1, L2, t2, t3:

Spe11uL
EcoR11uL
plasmid4uL
Buffer2uL
water12uL

37 ℃ 4 hour

Gel electrophoresis:
Products of double digest of L1, L2, t2, t3,
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
Loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 15min
Lane1: t2: insert 700bp;
Lane2: t3: insert 700bp;
Lane3: marker;
Lane5: L1: insert 1.1kb;
Lane6: L2: insert 1.1kb;
PKU 20090710 Shuke Wu 2.JPG

DNA Gel purification:
I made a big mistake here. I purified the brightest ones, which are vectors. So I do the double digest again.

Double digest (again):
L1, L2, t2, t3:

Spe11uL
EcoR11uL
plasmid4uL
Buffer2uL
water12uL

37 ℃ over night.

2009.7.11

Gel electrophoresis (again):
Products of double digest of L1, L2, t2, t3,
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
Loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 15min
Lane1: L1: insert 1.1kb;
Lane2: L2: insert 1.1kb;
Lane3: marker;
Lane5: t2: insert 700bp;
Lane6: t3: insert 700bp;
PKU 20090711 Shuke Wu 1.JPG

DNA ligation:

System10uL
Insert6uL
vector2uL
buffer1uL
T4 DNA ligase1uL

16℃ 4 hour
Insert: L1 L2 t2 t3;
Vector: terminator digested by EcoR1 and Xba1 (provided by Haoqian Zhang & Guosheng Zhang)

Transformation: (by Min Lin)
Products of ligation, competent cells 50uL each,
Smear to LB plate with Amp

2009.7.12

Every plate is very well: more than 100 clones
PCR:
14 tubes: L1×3+L2×3+t2×3+t3×3 and 2 negative controls
Master mix 5ul each, primer (standard primer) 0.5uL each, template;

Gel electrophoresis:
Products of PCR
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 15min
PKU 20090712 Shuke Wu 1.JPG
Lane 1: t2-1;
Lane 2~4: t3-3~1;
Lane 5: t2-3;
Lane 7: t2-1+2;
Lane 8: negative control1;
Lane 9: marker;
Lane 10: negative control2;
Lane 11~13: L2-3~1;
Lane 16~18: L1-3~1;

Result

There is a polluted line at about 1kb place, but it did not confuse us. The right place of L1 & L2 is about 1.3kb and of t2 & t3 is about 800bp.
L1-1~3, L2-1~3, t2-1&3 and t3-1~2 should be the positive clones.
4 clones were successfully constructed:
L1 & L2: 2×B0034-C0012-B0015
T2 & t3: 2×B0034-C0040-B0015

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