Team:Paris/August 4


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PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants n°L100 for screening PCR

Protocol of screening PCR

  • Mix
Name Vol (µl) Concentration
Quick Load 25µl 2X
OligoF_VF2 (O18) 1µl 10µM
OligoR_VR (O19) 1µl 10µM
water 23µl

  • 50µl of Mix PCR by tube/clone
  • one toothpick of each clone's colony by tube
  • Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

  • 10µl of ladder 1 kb
  • 20µl of screening PCR
  • migration ~30min at 100W on 0,8% gel

Results for L100

L100= D110 + D130 = RBS-tetR-ECFP-Ter Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png

Band 1-->8
Expected size 1042 pb
Measured size 1100 pb
Gel : L100(1-8)

==> Conclusion : we obtain a low quantity of PCR products, but at the good size.
So now, we know that we can amplify the clones that we want for L100 transformants bacteria.