User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/18

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August 18th, 2010

1. Make colony PCR to verify 1) L1: p30-MinBP + GFP E0240 (6 colonies); 2) L2: p30-MinBP + GFP BBa_K145015 (74 min) (10 colonies); 3) L4: Backbone pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min) (3 colonies).

  • Tubes are marked as follows:
-2.1-2.10 -> L2: p30-MinBP + GFP BBa_K145015 (74 min) (10 colonies)
-4.1-4.3 -> L4: Backbone pSB3K3 + Blue Promoter BBa_K238013 + GFP BBa_K145015 (74 min) (3 colonies)
  • 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. 30 cycles
  • 95ºC 45 seg
  • 55ºC 45 seg
  • 72ºC 1.5 min
3. 72ºC 10 min
4. Hold 4ºC


2. Run gel to verify PCR made on August 13th and 18th.

  • FAILED!!!



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