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INSA-Lyon/28 September 2010
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Latest revision as of 20:04, 25 October 2010
September 28th
Agarose gel electrophoresis for weight verification of 1F/24A/14K : none of the extraction contain 14K
Ligation with Ozyme protocol of 1F/24A and 14K in tet backbones
Ligation overnight of 1F/24A/14K
liquid culture of : blue, 8C/14K, L2/1N, 18A, phasin, phasin-intein, 24A and 14K
3 plasmid extractions (kit MN): Curli/22B-Tet; L2/1N-Cm; 18A-Amp. Samples are stored at -20°C.
Measures of nucleic acids concentrations with nanodrop (on 1,5µl extractions):
- Genomic DNA PHL818: 13ng/µl
- ompR PCR XT gel: 17,2ng/µl
- pSB1C3 Cm PCR XT gel: 4,6ng/µl
- pSB1T3 Tet PCR XT gel: 9,7ng/µl
- plasmid DNA L2/1N: 110ng/µl
- plasmid DNA Curli/22B: 12,5ng/µl
- plasmid DNA 18A: 31,7ng/µl
Conclusion: Not enough DNA (except for L2/1N)
Liquid culture Curli M.Gene in LB-Amp (from petri plate of 8/09) during the day, for plasmid extraction (kit MN), then DNA extraction was stored at -20°C.
Cloning ompR in pSB1C3: Digestion on 22µl of each DNA with 1µl EcoRI and 1µl PstI (Fermentas), in 6µl 10X buffer O during 1h at 37°C (thermomixer), then 20mn at 80°C (thermoinactivation, dry bath)
Ligation with T4 DNA Ligase (Fermentas): 30µl ompR E+P; 30µl pSB1C3 E+P; 7µl ligation buffer 10X; 1µl ligase; 2µl sterile water; at 15°C (thermomixer, no shaking), overnight
Liquid culture Curli M.Gene in LB-Amp (from petri plate of 8/09) for collection: at 37°C overnight
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