User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/09

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August 9th, 2010

1. Plasmid extraction to BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040) strain incubated on August 8th.

  • Plasmid extraction was done using the High Pure Plasmid Isolation Kit Roche.


2. 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 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 1-2:
1. BBa_I20260 RBS-GFP FWD-J23101 REV
2. Positive control: BBa_K137019 (~2855 bp) using primers Preffix FWD-Suffix REV.
  • 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 -> 32
DNA -> 2
Total volume -> 50
Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
  • 95ºC 45 seg
  • 60ºC 45 seg
  • 72ºC 1.5 min
3. 72ºC 5 min
4. Hold 4ºC