Paris/July 14

From 2007.igem.org

(Difference between revisions)
(→What to do ?)
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* Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
* Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
** Isolation of 10 different colonies on LB Citrate Erythro DAP plates
** Isolation of 10 different colonies on LB Citrate Erythro DAP plates
 +
 +
== PCRs ==
 +
 +
{{Paris_PCR| Title = DGAT amplification
 +
|Name= Lox71-FtsA-FtsZ-1
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 10µM 2.5µL
 +
|n_oligoR= 4 DEcoR1-FtsZ-R
 +
|v_oligoR= 10µM 2.5µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= toughpick in glycerol stock of MG1655
 +
|}}
 +
 +
 +
{{Paris_PCR| Title = FtsZ-2
 +
|Name= Lox71-FtsA-FtsZ1
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 5 DEcoR1-FtsZ-F
 +
|v_oligoF= 10µM 2.5µL
 +
|n_oligoR= 2 FtsZ-R
 +
|v_oligoR= 10µM 2.5µL
 +
|water= 34µL
 +
|pol= Phusion 0.5µL
 +
|DNA= toughpick in glycerol stock of MG1655
 +
|}}
 +
 +
 +
{{Paris_PCR| Title = DGAT amplification
 +
|Name= Lox66-DapAColi
 +
|Annealing= 55°C
 +
|Elongation= 2m00'
 +
|Cycles= 30
 +
|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= toughpick in glycerol stock of MG1655
 +
|}}

Revision as of 16:25, 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 : DGAT amplification
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 Lox71-FtsA-FtsZ1
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 : DGAT amplification
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
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