User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/13

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August 13th, 2010

1. Repeat 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).
  • I will use different volumes of DNA (0.1ul, 0.25ul, 0.5ul, 0.75ul, 1ul)
  • Tubes are marked 1-6:
1. BBa_I20260 RBS-GFP FWD-J23101 REV (0.1ul DNA)
2. BBa_I20260 RBS-GFP FWD-J23101 REV (0.25ul DNA)
3. BBa_I20260 RBS-GFP FWD-J23101 REV (0.5ul DNA)
4. BBa_I20260 RBS-GFP FWD-J23101 REV (0.75ul DNA)
5. BBa_I20260 RBS-GFP FWD-J23101 REV (1ul DNA)
6. BBa_I20260 Suffix FWD-J23101 REV (1ul DNA)
PCR with Platinum Taq DNA polymerase (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 2.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.4
Template DNA -> 2
HPLC -> 35.1
Total volume -> 50
  • 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