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:
| Spe1 | 1uL
|
| Pst1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
lacI, araC:
| Xba1 | 1uL
|
| Pst1 | 1uL
|
| plasmid | 4uL
|
| Buffer | 2uL
|
| water | 12uL
|
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:
| System | 10uL
|
| Insert | 4uL
|
| vector | 1uL
|
| water | 3uL
|
| buffer | 1uL
|
| T4 DNA ligase | 1uL
|
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.
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