User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/07/03

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July 3rd, 2010

1. Inactivate enzyme from ligation made on July 2nd. Incubate 10 min at 65ºC.

2. Make transformations with ligations made on July 2nd.

1. Dephosphated p30-MinBP SpeI-PstI + GFP E0240 XbaI-PstI.
4. Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction + Blue Promoter EcoRI-SpeI restriction + GFP BBa_K145015 XbaI-PstI restriction.
5. Dephosphated plasmid 18 XbaI-PstI restriction + GFP BBa_K145015 XbaI-PstI restriction.
  • 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.

3. Prepare culture dishes with Amp 100, procedure explained on April 20th.

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