Paris/July 18

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[[Paris/July 17|yesterday]] -- [[Paris/July 19|tomorrow]] <br>
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== Transduction of w121 strain ==
 +
Titration of the stock of phage from 12.7.7. : ~10<sup>9</sup> phages/ml : OK
 +
<br>
== FtsZ<sup>TS</sup> strain screening ==
== FtsZ<sup>TS</sup> strain screening ==
We want to test these strains :
We want to test these strains :
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== Minipreps==
== Minipreps==
We have 2 clones for each plasmid :
We have 2 clones for each plasmid :
-
<br>
 
* E0422
* E0422
* E0241
* E0241
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* B0030
* B0030
* J61047
* J61047
-
<br>
+
Elution within 50µL H2O<br>
-
DNA is within the Miniprep box in the -20°C fridge
+
DNA is within the Miniprep box in the -20°C fridge.
 +
 
 +
==Digestion products==
 +
Photos !!!
 +
== DAP solution contamination test ==
 +
Results :
 +
* 50µL of H<sub>2</sub>0 (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==
 +
 
 +
{{Paris_PCR_0| Title = Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2
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|Annealing= 50, 55, 60, 65°C
 +
|Elongation= 3m00'
 +
|Cycles= 35
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 2.5µL
 +
|n_oligoR= 2  FtsZ-R
 +
|v_oligoR= 2.5µL
 +
|water= 30µL
 +
|pol= Phusion 0.5µL
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|DNA= Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL
 +
|Size=2580
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|Success=NO
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|Image=
 +
|Band=
 +
|}}
 +
 
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== E.coli pKS::DGAT ==
 +
 
 +
* After overnight culture (see [[Paris/July_17#Exponential_culture_of_E.coli_pKS::DGAT_and_microscopy|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 [[Paris/PROTOCOLS#Solid_M9_Minimum_Medium|Protocols]]) replacing lactose/galactose by 2mM oleate, too.

Latest revision as of 17:48, 7 October 2007

yesterday -- tomorrow

Contents

Transduction of w121 strain

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

FtsZTS 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.