User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/11

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August 11th, 2010

1. Make PCR to change RBS into BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), this because we have one construction with this plasmid + Blue Promoter + GFP E0240, and we want to have both GFP’s under the same RBS, so we can characterize better the promoters without noise caused if we used different Ribosome Binding Sites.

  • PCR will be done with Platinum Taq Polymerase.
  • I will use primers RBS_GFP FWD-J23101 REV
  • PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
  • Tubes are marked 1-3:
1. BBa_I20260 RBS-GFP FWD-J23101 REV.
2. BBa_I20260 Suffix FWD-J23101 REV.
3. Positive control: L1 Preffix FWD-Suffix REV.
  • PCR with Platinum Taq DNA polymerase
Reactive (ul x sample)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 2.5
Primer mix (10uM each) -> 2
Platinum Taq DNA Pol -> 0.4
Template DNA -> 4
HPLC -> 35.1
Total volume -> 50
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 60ºC 45 seg
  • 72ºC 4 min
3. 72ºC 10 min
4. Hold 4ºC



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