User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/04/22

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April 22nd, 2010

1. Transformación con plásmido pSB1A2 y pSB1AK3 (control +).

  • Extract BBa_E0430 DNA from 2009 Distribution Kit Plate, suspend in 15ul of HPLC, transfer to a new eppendorf tube.
  • Plasmid pSB1AK3 (3189bp) was used as positive control, contains the biopart B0015 (129 bp).
Transformation method:
-Unfreeze competent cells (keep on ice).
-Add 5ul of exogenous DNA to 100ul of compentent cells.
-Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
-Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
-Incubate 5 min on ice.
-Transfer the cells to a tube with 1ml of LB medium
-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).
-Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
-Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
-Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
-Incubate the plates at 37ºC overnight.