Molecular cloning: RBS-lacI/tetR + terminator
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:
| Spe1 | 1uL
|
| EcoR1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
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;
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:
| Spe1 | 1uL
|
| EcoR1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
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;
DNA ligation:
| System | 10uL
|
| Insert | 6uL
|
| vector | 2uL
|
| buffer | 1uL
|
| T4 DNA ligase | 1uL
|
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
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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