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

Notebook > AND Gate 1 > Input > Molecular cloning: 6×RBS+ araC/ lacI

Molecular cloning: 6×RBS+ araC/ lacI


Resource: 6 RBS: from the parts: B0030, B0032, B0034, J61100, J61107, J61127; renamed as RBS1 RBS2…RBS6; araC: C0080 lacI: C0012

2009.7.6
Plasmid mini prep: 6 RBS; lacI; araC;

Double digest: 6 RBS: lacI, araC: 37 ℃ 4 hour

2009.7.7
Gel electrophoresis: Products of double digest of lacI and araC, marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× voltage and time: 60V 5min; 120V 15min lane1: lacI plasmid; lane2: araC plasmid; lane3: marker; lane4: digested product of lacI; lane5: digested product of araC;

DNA Gel purification: lacI and araC

PCR product purification: 6 RBS

DNA ligation: 16℃ 4 hour Insert: lacI; araC; tetR (from Min Lin); Vertor: 6 RBS

Transformation:  Products of ligation, competent cells 50uL each, Smear to LB plate with Amp

2009.7.8
Every plate is very well: more than 100 clones Waiting for PCR to check the positive clones

2009.7.9
PCR: Master mix 5ul, primer (standard primer) 0.5uL each, template;

2009.7.10
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 Up row: lane 1~3: tetR+RBS1 1~3; lane 4~6: tetR+RBS2 1~3; lane 7~9: tetR+RBS3 1~3; lane 10: marker; lane 11~12: tetR+RBS4 2~3; lane 13~15: tetR+RBS5 1~3; lane 16~18: tetR+RBS6 1~3; down row: lane 1~3: lacI+RBS1 1~3; lane 4~6: lacI+RBS2 1~3; lane 7~9: lacI+RBS3 1~3; lane 10: marker; lane 11~12: lacI+RBS4 2~3; lane 13~15: lacI+RBS5 1~3; lane 16~18: lacI+RBS6 1~3;

Result
10 clones were successfully constructed: RBS1~5+lacI and tetR; 2 clones were failed: RBS6+lacI and tetR.