Paris/July 14
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David.bikard (Talk | contribs) (→PCRs) |
Eismoustique (Talk | contribs) (→What has been done) |
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* Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121): | * Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121): | ||
** Isolation of 10 different colonies on LB Citrate Erythro DAP plates | ** Isolation of 10 different colonies on LB Citrate Erythro DAP plates | ||
| + | |||
| + | |||
| + | * Seeding LB-Amp cultures of the following strains | ||
| + | ** DH5alpha transformed with the plasmid carrying the Biobrick pJ23100 | ||
| + | ** DH5alpha transformed with the plasmid carrying the Biobrick B0015 | ||
| + | |||
| + | these two ON cultures will allow us to perform (tomorrow): | ||
| + | ** Minipreps to isolate the plasmids carrying the biobricks | ||
| + | ** Glycerol stocks of the transformed strains | ||
== PCRs == | == PCRs == | ||
Revision as of 16:13, 15 July 2007
What to do ?
- Remove the plate of the transduction experiment and the transformation ones from the 37°C incubator (Upper-left shelf): If it has worked and if someones comes on Sunday:
- isolation of the clones from the transduction on petri-dishes (LB+DAP+Erm+Citrate)
- LB + antibio culture of the transformants (to make a glycerol stock and to do MiniPreps)
- Make a gel (0.8%) and migrate the PCR products
- Purify them
- Do the assembly PCR
If enough time (we can also wait to have the plasmids to do everything at the same time):
- Digestion of the purifications products with appropriate enzymes
- Purification
What has been done
- Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
- Isolation of 10 different colonies on LB Citrate Erythro DAP plates
- Seeding LB-Amp cultures of the following strains
- DH5alpha transformed with the plasmid carrying the Biobrick pJ23100
- DH5alpha transformed with the plasmid carrying the Biobrick B0015
these two ON cultures will allow us to perform (tomorrow):
- Minipreps to isolate the plasmids carrying the biobricks
- Glycerol stocks of the transformed strains
PCRs
| PCR : Lox71-FtsA-FtsZ-1 | ||
|---|---|---|
| Name | Lox71-FtsA-FtsZ-1 | |
| Annealing T° | 55°C | |
| Time of elongation | 2m00' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 3 Lox71-FtsA-F | 10µM 2.5µL |
| oligoR | 4 DEcoR1-FtsZ-R | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | toughpick in glycerol stock of MG1655 | |
| PCR : FtsZ-2 | ||
|---|---|---|
| Name | FtsZ-2 | |
| Annealing T° | 55°C | |
| Time of elongation | 2m00' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 5 DEcoR1-FtsZ-F | 10µM 2.5µL |
| oligoR | 2 FtsZ-R | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | toughpick in glycerol stock of MG1655 | |
| PCR : Lox66-DapAColi | ||
|---|---|---|
| Name | Lox66-DapAColi | |
| Annealing T° | 55°C | |
| Time of elongation | 2m00' | |
| Number of Cycles | 30 | |
| Buffer | 5x 10µL | |
| MgCl2 | 10µM 0µL | |
| dNTP | 10µM 1µL | |
| oligoF | 6 Lox66-DapAColi-F | 10µM 2.5µL |
| oligoR | 7 DapAColi-R | 10µM 2.5µL |
| water | 34µL | |
| polymerase | Phusion 0.5µL | |
| DNA | toughpick in glycerol stock of MG1655 | |
