IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/29

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LovTAP fused to promoters and Lumazine:Ligations to backbone pSB3K3 and pSB1C3

I'm working in Step 7 of the Repeated experiment:Working on LovTAP fused to promoters and Lumazine:Ligations to backbone pSB3K3 and 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 fused to promoters and Lumazine, if the ligations were correctly done.

Results:LovTAP fused to promoters and Lumazine:Ligations to backbones pSB3K3 and pSB1C3.Procedure Step 7

  • Colonies selected to do the colony PCR reactions:

Ligations to plasmid pSB3K3

LovTAP + J23117 isolated from colonies 2, 3 and 4.

LovTAP + J23105 isolated from colonies 1, 2 and 3.

LovTAP + J23114 isolated from colonies 9,13 and 14.

LovTAP + J23102 isolated from colonies 1,11 and 13.


Ligations to plasmid pSB1C3

LovTAP + J23117 isolated from colonies 1,6 and 7.

LovTAP + J23105 isolated from colonies 1, 12 and 13.

LovTAP + J23114 isolated from colonies 1,2 and 3.

LovTAP + J23102 isolated from colonies 1,2 and 8.

Lumazine (Mr.Gene) colonies 1 and 2.


Colony PCRs: LovTAP fused to promoters and Lumazine.Ligations to backbones pSB3K3 and pSB1C3.Lane1:Ladder. Lane 2,3 and 4: PCR amplified product LovTAP + J23114 + pSB3K3 from colonies 9,13 and 14 respectively.Lane 5,6 and 7:PCR amplified product LovTAP + J23114 + pSB1C3 isolated from colonies 1,2 and 3, respectively. Lane 8,9 and 10:PCR amplified product LovTAP + J23102 + pSB3K3 isolated from colonies 1,11 and 13, respectively. Lane 14 and 15:PCR amplified product LovTAP + J23117 + pSB3K3 isolated from colonies 2 and 3 , respectively. The other lanes are samples from other experiments.


Colony PCRs: LovTAP fused to promoters and Lumazine.Ligations to backbones pSB3K3 and pSB1C3.Lane1:Ladder. Lane 2: PCR amplified product LovTAP + J23117 + pSB3K3 isolated from colony 4.Lane 3,4 and 5: PCR amplified product LovTAP + J23105+pSB1C3 isolated from colonies 1, 12 and 13, respectively. Lane 9,10 and 11:PCR amplified product LovTAP + J23102 + pSB1C3 isolated from colonies 1,2 and 8, respectively. Lane 12,13and 14:PCR amplified product LovTAP + J23105+ pSB3K3 isolated from colonies 1, 2 and 3 , respectively. Lane 15: PCR amplified product LovTAP + J23117 +pSB1C3 from colony 1.The other lanes are samples from other experiments.
Colony PCRs: LovTAP fused to promoters and Lumazine.Ligations to backbones pSB3K3 and pSB1C3.Lane1:Ladder. Lane 2 and 3: PCR amplified product LovTAP + J23117 +pSB1C3 from colonies 6 and 7.Lane 4 and 5: PCR amplified product Lumazine (Mr.gene)+pSB1C3 from colonies 1 and 2, respectively. The other lanes are samples from other experiments.