Paris/July 17
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Nicolas C. (Talk | contribs) (→PCRs) |
Nicolas C. (Talk | contribs) |
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|DNA= 1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2 | |DNA= 1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2 | ||
|}} | |}} | ||
| + | |||
| + | == DAP solution contamination test == | ||
| + | We spread on LB plates DAP solution : | ||
| + | * 50µL of aliquot DAP 50mM (in use) | ||
| + | * 50µL of DAP 1mM | ||
| + | * 50µL of DAP 0.2 mM | ||
| + | * 50µL of DAP 50mM | ||
Revision as of 11:52, 17 July 2007
PCRs
- The assembly PCRs did not work... we'll try them again !
- The Lox66-DapAColi PCRs worked very well in all conditions
=> Gel purification
| PCR : FtsZ-E.Coli | ||
|---|---|---|
| Name | Ftsz-E.Coli | |
| Annealing T° | 55°C | |
| Time of elongation | 2m30' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 1 Lox71-FtsZ-F | 10µM 2.5µL |
| oligoR | 2 FtsZ-R | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | toothpick in glycerol stock of MG1655 | |
| PCR : DapA-Subtilis | ||
|---|---|---|
| Name | DapA-Subtilis | |
| Annealing T° | 55°C | |
| Time of elongation | 2m30' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 8 Lox66-DapASubtilis-F | 10µM 2.5µL |
| oligoR | 9 DapASubtilis R | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | 0.5µL of Subtilis DNA from stock (DB tube in fridge) | |
| PCR : Assembling PCR DGAT | ||
|---|---|---|
| Name | assembly PCR of DGAT | |
| Annealing T° | 55°C | |
| Time of elongation | 2m30' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 10µM 2.5µL | |
| oligoR | 10µM 2.5µL | |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | 1µL of PCR product DGAT 1 1µL of PCR product DGAT 2 | |
| PCR : Assembling PCR FtsZ-FtsA | ||
|---|---|---|
| Name | assembly PCR of FtsZ-FstA | |
| Annealing T° | 55°C | |
| Time of elongation | 2m30' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 3 | 10µM 2.5µL |
| oligoR | 2 | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | 1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2 | |
DAP solution contamination test
We spread on LB plates DAP solution :
- 50µL of aliquot DAP 50mM (in use)
- 50µL of DAP 1mM
- 50µL of DAP 0.2 mM
- 50µL of DAP 50mM
