User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/08/24

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August 24th, 2010

1. Inactivate ligation enzyme, 10 min at 65ºC.

2. Transform competent E. coli K12 cells with ligation 2: p30-MinBP + GFP BBa_K145015 (74 min).

  • Transformation method:
  1. Unfreeze competent cells (keep on ice).
  2. Add 5ul of exogenous DNA to 100ul of compentent cells.
  3. Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
  4. Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
  5. Incubate 5 min on ice.
  6. Transfer the cells to a tube with 1ml of LB medium
  7. Incubate the cells in the LB medium for 1hr at 37ºC and shaking (during this period the cells recover their metabolic activity, replicate and synthesize the proteins encoded in the material just inserted).
  8. Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
  9. Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
  10. Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
  11. Incubate the plates at 37ºC overnight.

3. 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 make 2 reactions with primers RBS-GFP FWD-J23101 REV (Tubes 1-2).
  • PCR product is expected to be 3670 bp (941 biopart + 2729 primer pSB3K3).
  • Control: Preffix FWD-Suffix REV (Tube 3).
  • Control: Suffix FWD-J23101 REV (Tube 4).
  • Control: RBS-GFP FWD-Suffix REV (Tube 5).
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