User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/05

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August 5th, 2010

1. Prepare culture dishes with TOP medium.

2. Plate Vibrio Fischeri MJ11 on solid BOSS medium and TOP medium.

3. Make PCR to verify constructions L2 and L4, and verify length of the inserts, specially GFP BBa_K145015 (74 min).

  • PCR will be done with Taq Polymerase.
  • I will use primers RBS-GFP FWD-Suffix REV; Blue Promoter FWD-Suffix REV; Preffix FWD-Suffix REV.
  • Tubes are marked 1-6:
1. L2 RBS-GFP FWD-Suffix REV
2. L2 Blue Promoter FWD-Suffix REV
3. L2 Preffix FWD-Suffix REV
4. L4 RBS-GFP FWD-Suffix REV
5. L4 Blue Promoter FWD-Suffix REV
6. L4 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
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