User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/02

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August 2nd, 2010

1. Verify constructions by Colony PCR to amplify inserts from the following ligations:

  • L1: p30-MinBP + GFP E0240
  • L2: p30-MinBP + GFP BBa_K145015 (74 min)
  • L4: Backbone plasmid pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min)
  • L5: Plasmid 18 pSB1T3 + GFP BBa_K145015
  • L6: Religation p30-MinBP to make it a standard BioBrick


  • Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:
PCR with Taq DNA polymerase
Reactives (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. 30 cycles
  • 95ºC 45 seg
  • 60ºC 45 seg
  • 72ºC 1.5 min
3. 72ºC 5 min
4. Hold 4ºC


2. Run gel to verify Colony PCR.

Colony PCR to verify constructions 2Aug2010.JPG

Lanes: 1) Green ladder; 2) L1: p30-MinBP + GFP E0240; 3) L2: p30-MinBP + GFP BBa_K145015 (74 min); 4) L4: Backbone plasmid pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min); 5) L5: Plasmid 18 pSB1T3 + GFP BBa_K145015; 6) L6: Religation p30-MinBP to make it a standard BioBrick; 7) BBa_I20260 pSB3K3 + J23101 + GFP E0040