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Revision as of 17:37, 16 July 2007 (view source) David.bikard (Talk | contribs) (→PCRs) ← Older edit		Revision as of 20:48, 16 July 2007 (view source) Eismoustique (Talk | contribs) (→PCRs) Newer edit →
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	* Lox71-FtsA-FtsZ-1 + FtsZ-2		* Lox71-FtsA-FtsZ-1 + FtsZ-2
	* DGAT-1 + DGAT-2		* DGAT-1 + DGAT-2
		+
		+
		+	The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures
		+
		+	{{Paris_PCR| Title = Lox66-DapAColi
		+	|Name= Lox66-DapAColi
		+	|Annealing= 50-65°C
		+	|Elongation= 2m00'
		+	|Cycles= 35
		+	|Buffer=  5x 10µL
		+	|MgCl2= 10µM 0µL
		+	|dNTP= 10µM 1µL
		+	|n_oligoF= 6 Lox66-DapAColi-F
		+	|v_oligoF= 10µM 2.5µL
		+	|n_oligoR= 7 DapAColi-R
		+	|v_oligoR= 10µM 2.5µL
		+	|water= 34µL
		+	|pol= Phusion 0.5µL
		+	|DNA= toothpick in glycerol stock of MG1655
		+	|}}

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Revision as of 20:48, 16 July 2007

Contents 1 Plasmid pKs::DGAT expression in E. Coli 2 Growth kinetics of w121 strain 3 Transduction of MG1655 with P1 stock made on w121 4 MiniPreps 5 PCR purification 6 PCRs

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 n^th 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
Name	Lox66-DapAColi
Annealing T°	50-65°C
Time of elongation	2m00'
Number of Cycles	35
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	toothpick in glycerol stock of MG1655