From 2007.igem.org

Revision as of 15:13, 30 July 2007 (view source) Davidpz (Talk | contribs) (→E.coli pKS::DGAT) ← Older edit		Latest revision as of 17:48, 7 October 2007 (view source) Nicolas C. (Talk | contribs)
Line 1:		Line 1:
		+	[[Paris/July 17|yesterday]] -- [[Paris/July 19|tomorrow]] <br>
	== Transduction of w121 strain ==		== Transduction of w121 strain ==
	Titration of the stock of phage from 12.7.7. : ~10<sup>9</sup> phages/ml : OK		Titration of the stock of phage from 12.7.7. : ~10<sup>9</sup> phages/ml : OK

---

Latest revision as of 17:48, 7 October 2007

yesterday -- tomorrow

Contents 1 Transduction of w121 strain 2 FtsZTS strain screening 3 Minipreps 4 Digestion products 5 DAP solution contamination test 6 PCRs 7 E.coli pKS::DGAT

Transduction of w121 strain

Titration of the stock of phage from 12.7.7. : ~10⁹ phages/ml : OK

FtsZ^TS strain screening

We want to test these strains :

• 121.4
• 121.5
• 121.6
• 129
• 1.129
• DCR 14.1
• DCR 14.2
• DCR 14.3

I made glycerol stock of these strains, stored in the freezer. Test :

• Spread these strains on two plates (preheated before spreading) :
  • One at 30°C
  • One at 42°C

The results tomorrow.

Minipreps

We have 2 clones for each plasmid :

• E0422
• E0241
• E0840
• B0030
• J61047

Elution within 50µL H2O
DNA is within the Miniprep box in the -20°C fridge.

Digestion products

Photos !!!

DAP solution contamination test

Results :

• 50µL of H₂0 (control) OK
• 50µL of aliquot DAP 50mM (in use) OK
• 50µL of DAP 1mM CONTAMINATED
• 50µL of DAP 0.2 mM OK
• 50µL of DAP 50mM OK

PCRs

PCR : Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2580
50, 55, 60, 65°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL		NO
3m00'		Oligo R 10µM	2 FtsZ-R	2.5µL		Image (click to enlarge)
Number cycles		Water	30µL			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL

E.coli pKS::DGAT

• After overnight culture (see July 17), we do not observe significant triglyceride synthesis.
• We decide to test in solid culture and wait some days. E.coli transformed by pKS::DGAT and the negative control (E.coli transformed by part B0015) were spread on LB medium +- oleate 2mM +- IPTG (0.4mM).
• We try in M9 Minimum Medium (see Protocols) replacing lactose/galactose by 2mM oleate, too.