IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/11

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Ligation Procedure:LovTAP from Mr.Gene to plasmid pSB1C3

I'm working in Step 7 of the Ligation Procedure: LovTAP_Promoters and BBa_K098991 to plasmid pSB1C3.

  • 7.Colony PCR to confirm ligations:

Analyze the colonies with Colony PCR to confirm that they contain the correct ligation.

The primers that I am using are:

Forward (5'->3'): Preffix primer.

Reverse:(5'->3'): Suffix primer.

These primers would amplify LovTAP, if the ligation was correctly done.

Results:Ligation Procedure Step 7

After re-culturing the colonies transformed with each ligation mixture, only colonies 2, 5, 6 and 10 harboring the LovTAP amplified product from Mr.Gene fused to plasmid pSB1C3 grew correctly.

Thus, these colonies were selected to do the colony PCR reactions.

Colony PCR Ligation LovTAP (Mr.Gene) to plasmid pSB1C3..Lane1:Ladder. Lane 5,6,7 and 8:LovTAP from Mr.Gene+ pSB1C3(colonies 2,5,6 and 10 respectively).The other lanes are samples from other experiments.