IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/08

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Ligation Procedure:LovTAP-Promoters and BBa_K098991 to plasmid pSB3K3

I'm working in Step 7 of the Ligation Procedure: LovTAP_Promoters and BBa_K098991 to plasmid pSB3K3.

  • 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 ligated to each promoter and BBa_K098991, if the ligation was correctly done.

Results:Ligations LovTAP-Promoters with plasmid pSB3K3.Procedure Step 7

After re-culturing the colonies transformed with each ligation mixture, only colonies harboring the LovTAP+J23105 (6 and 7) and LovTAP+J23114 (3) fused to plasmid pSB3K3 grew correctly.

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

Colony PCRs Ligations LovTAP+promoters to plasmid pSB3K3..Lane1:Ladder. Lane2:Plasmid LovTAP-J23114 + pSB3K3 (colony 3). Lane3 and 4:Plasmid LovTAP-J23105 + pSB3K3 (colonies 6 and 7, respectively).
Colony PCRs Ligations LovTAP+promoters to plasmid pSB3K3..Lane1:Ladder. Lane2:Plasmid LovTAP-J23114 + pSB3K3 (colony 3). Lane3 and 4:Plasmid LovTAP-J23105 + pSB3K3 (colonies 6 and 7, respectively).

Results:Ligation. Part BBa_K098991 with plasmid pSB3K3.Procedure Step 7

After re-culturing the colonies transformed with the ligation mixture, only one colony (1) harboring the Part BBa_K098991(cI regulated promoter+RBS+GFP 933bp) fused to plasmid pSB3K3 grew correctly.

Thus, this colony was selected to do the colony PCR reaction.

Colony PCRs Ligations LovTAP+promoters to plasmid pSB3K3..Lane1:Ladder. Lane15:Colony PCR Part BBa_K098991 amplified product fused to plasmid pSB3K3 from colony 1. The other lanes are samples from other experiments
Colony PCRs Ligations LovTAP+promoters to plasmid pSB3K3..Lane1:Ladder. Lane15:Colony PCR Part BBa_K098991 amplified product fused to plasmid pSB3K3 from colony 1. The other lanes are samples from other experiments



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