User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/05

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October 5th, 2010

1. Make colony PCR of the following strains:

  • pSB1A2 + J23101 (SpeI-PstI restriction) + Δ RBS + GFP E0040: Colonies 1 (tube 1, T1), 3 (T2), 5(T3).
  • Religation of pSB3K3 + J23101 (SpeI restriction): 10 colonies (tubes 4-13)
  • Religation of pSB4A5 (p30) + Minimmum Blue Promoter: Colonies 1-3 (T14-T16), 5 (T17), 7-10 (T18-T21).
  • 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 1 min
3. 72ºC 10 min
4. Hold 4ºC