IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Input/2009/07/10/Shuke Wu

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.

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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.