Paris/July 14

From 2007.igem.org

(Difference between revisions)
(PCRs)
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== PCRs ==
== PCRs ==
-
{{Paris_PCR| Title = DGAT amplification
+
{{Paris_PCR| Title = Lox71-FtsA-FtsZ-1
|Name= Lox71-FtsA-FtsZ-1  
|Name= Lox71-FtsA-FtsZ-1  
|Annealing= 55°C
|Annealing= 55°C
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{{Paris_PCR| Title = FtsZ-2
{{Paris_PCR| Title = FtsZ-2
-
|Name= Lox71-FtsA-FtsZ1
+
|Name= FtsZ-2
|Annealing= 55°C
|Annealing= 55°C
|Elongation= 2m00'
|Elongation= 2m00'
Line 57: Line 57:
-
{{Paris_PCR| Title = DGAT amplification
+
{{Paris_PCR| Title = Lox66-DapAColi
|Name= Lox66-DapAColi
|Name= Lox66-DapAColi
|Annealing= 55°C
|Annealing= 55°C

Revision as of 16:27, 14 July 2007

What to do ?

  • Remove the plate of the transduction experiment and the transformation ones from the 37°C incubator (Upper-left shelf): If it has worked and if someones comes on Sunday:
    • isolation of the clones from the transduction on petri-dishes (LB+DAP+Erm+Citrate)
    • LB + antibio culture of the transformants (to make a glycerol stock and to do MiniPreps)
  • Make a gel (0.8%) and migrate the PCR products
  • Purify them
  • Do the assembly PCR

If enough time (we can also wait to have the plasmids to do everything at the same time):

  • Digestion of the purifications products with appropriate enzymes
  • Purification


What has been done

  • Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
    • Isolation of 10 different colonies on LB Citrate Erythro DAP plates

PCRs

PCR : Lox71-FtsA-FtsZ-1
Name Lox71-FtsA-FtsZ-1
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 3 Lox71-FtsA-F 10µM 2.5µL
oligoR 4 DEcoR1-FtsZ-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toughpick in glycerol stock of MG1655


PCR : FtsZ-2
Name FtsZ-2
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 5 DEcoR1-FtsZ-F 10µM 2.5µL
oligoR 2 FtsZ-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toughpick in glycerol stock of MG1655


PCR : Lox66-DapAColi
Name Lox66-DapAColi
Annealing T° 55°C
Time of elongation 2m00'
Number of Cycles 30
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 toughpick in glycerol stock of MG1655