User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/13

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September 13th, 2010

1. PCR to amplify BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), one reaction will amplify the plasmid including the J23101 promoter and another reaction will amplify only the GFP E0040.

  • PCR will be done with Platinum Taq Polymerase.
  • I will make 2 reactions with primers Suffix FWD-J23101 REV (Tubes 1-2).
  • PCR product is expected to be 3670 bp (35 promoter + 2729 plasmid pSB3K3).
  • To amplify the GFP E0040: RBS GFP FWD-Suffix REV (Tubes 3-4) GFP length is 720 bp.
PCR with Platinum Taq DNA polymerase Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 1.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.4
Template DNA -> 2
HPLC -> 35.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • 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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