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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: 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:

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: 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