Paris/July 17
From 2007.igem.org
Contents |
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 : Assembly 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 : Assembly 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 | |
I make a gel to check PCR products. To put on gel :
- 50µL of PCR product
- 5µL of "bleu de charge" jaune.
DAP solution contamination test
We spread on LB plates DAP solution :
- 50µL of H20 (control)
- 50µL of aliquot DAP 50mM (in use)
- 50µL of DAP 1mM
- 50µL of DAP 0.2 mM
- 50µL of DAP 50mM
+ filtration of the DAP stock
Transduction of MG1655 with P1 stock made on w121
As it still didn't work, we try for now with culture of MG1655 in exponential growth rate (ExpGR)
OD=0.6
- Preparation of samples:
- Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 20mn @ 37°C = adsorption
- Centrifugation 1mn @ 10000rpm
- Resuspension in LB + Na Citrate 20mM + DAP 300µM
- 1H growth @ 37°C
- Plate on LB + Na Citrate + Erythromycin + DAP
Transformations of Biobricks : results
Transformation of biobricks : we obtained hundreds of clones for each biobrick.
Transformation in DH5alpha subcloning efficiency
Spread on LB-Amp
- BBa_B0030 pSB1A2 : RBS (well 3G plate 1)
- BBa_E0422 pSB1A2 : ECFP (RBS+LVA+Term) (well 11G plate 1)
- BBa_E0241 pSB1A2 : PoPs to GFP converter (well 15c plate 2)
- BBa_E0840 gfp tri-part; strong rbs (well 16E plate 1)
- BBa_J61047 pSB1A2 : Cre ORF (well 8P plate 4)
