User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/29

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June 29th, 2010

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

2. Transformations with the ligations from June 28th:

  • Transformation 1: p30-MinBP SpeI-PstI restrictionn (2ul) + GFP E0240 XbaI-PstI restriction (5ul)
  • Transformation 2: p30-MinBP SpeI-PstI restriction (2ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)
  • Transformation 3: Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction (2ul) + Blue Promoter EcoRI-SpeI restriction (5ul) + GFP E0240 XbaI-PstI restriction (5ul)
  • Transformation 6: Religate p30-MinBP SpeI restriction (2ul)

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.