Paris/July 16
From 2007.igem.org
Nicolas C. (Talk | contribs) (→Transformations of Biobricks) |
Nicolas C. (Talk | contribs) (→PCRs) |
||
| Line 40: | Line 40: | ||
The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures | The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures | ||
| - | {{ | + | {{Paris_PCR_0| Title = Lox66-DapAColi |
|Name= Lox66-DapAColi | |Name= Lox66-DapAColi | ||
|Annealing= 50-65°C | |Annealing= 50-65°C | ||
| Line 55: | Line 55: | ||
|pol= Phusion 0.5µL | |pol= Phusion 0.5µL | ||
|DNA= toothpick in glycerol stock of MG1655 | |DNA= toothpick in glycerol stock of MG1655 | ||
| + | |Size= | ||
| + | |Success=YES | ||
| + | |Band= | ||
| + | |Image= | ||
|}} | |}} | ||
| - | |||
== Transformations of Biobricks == | == Transformations of Biobricks == | ||
Revision as of 17:29, 18 July 2007
Contents |
Plasmid pKs::DGAT expression in E. Coli
We tried to see TG with NR died on E. Coli cells transfected with pKs::DGAT, an IPTG-inducible promoter.
We tried different growth media containing more or less oleate, that should in theory increase TG synthesis.
Results : we don't see the inductible effect of IPTG. We can think that :
- Either the fluorescence without IPTG is due to a leak of the promoter
- Either DGAT is not induce in presence of IPTG, and the fluorescence we see is only a background.
Growth kinetics of w121 strain
Results of the previous day : we lost everything because of a crash of the computer :( Sorry Eimad !
Transduction of MG1655 with P1 stock made on w121
- Control (1mL LB MgSO4 30mM; CaCl2 15mM)
- 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
- 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
- 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
=>For the nth time, it is not working : we have only contaminants.
MiniPreps
- I0500 clones 1, 2
- pJ23107 clones 1, 2
PCR purification
- Lox71-FtsZ1
- FtsZ2
- DGAT1
- DGAT2
PCRs
The Lox66-DapAColi PCR did not work... We'll try again with different annealing temperature
Assembly PCRs:
- Lox71-FtsA-FtsZ-1 + FtsZ-2
- DGAT-1 + DGAT-2
The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures
| PCR : Lox66-DapAColi | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | ||||
| 50-65°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 6 Lox66-DapAColi-F | 10µM 2.5µL | YES | ||
| 2m00' | Oligo R 10µM | 7 DapAColi-R | 10µM 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µL | [[Image:|30px]] | |||
| 35 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | toothpick in glycerol stock of MG1655 | |||||
Transformations of Biobricks
As in the protocol given in the registry. Transformation of biobricks :
- 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)
Transformation in DH5alpha subcloning efficiency
Spread on LB-Amp
