User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/25

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September 25th, 2010

1. Make colony PCR of the ligations:

  • pSB3K3-J23101 + Δ RBS-GFP E0040 (3 colonies, tubes 1-3)
  • pSB3K3-Min. Blue Promoter + Δ RBS-GFP E0040 (1 colony, tube 4)

Colony PCR methods:

  1. Take a colony and resuspend in 200ul of Tri-EDTA 10/1-NaCl 10 mM.
  2. Heat 10 min at 95ºC.
  3. Centrifugue at 14000 rpm 2 min.
  4. Take 10 ul as template for PCR.
  • Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:

PCR with Taq DNA polymerase Reactive (ul x sample)

Taq Polymerase -> 1
Taq Reaction Buffer 10X -> 5
MgCl 50mM (can be used up to 3ul) -> 2.5
dNTP’s 0.4ug/ul -> 2.5
Primer Forward (can be used up to 3ul) -> 2.5
Primer Reverse (can be used up to 3ul) -> 2.5
HPLC -> 24
DNA -> 10
Total volume -> 50
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 55ºC 45 seg
  • 72ºC 4 min
3. 72ºC 10 min
4. Hold 4ºC



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