IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/30

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Ligation Procedure:Lumazine/LuxY with plasmid pSB1C3 and promoter J23101.

I'm working in Step 7 of the Ligation Procedure: Lumazine, LuxY and P0451 with plasmid pSB1C3 and promoter J23101.

  • 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 Lumazine, LuxY and P0451 ligated to each promoter, if the ligations were correctly done.

Results:Ligations Lumazine, LuxY and P0451 with plasmid pSB1C3 and promoter J23101.Procedure Step 7

  • Colonies selected to do the colony PCR reactions:

P0451 + J23101 ligation from colonies 1,5 and 23.

Colony PCRs:Ligations P0451 with and promoter J23101..Lane1:Ladder. Lane 11,12 and 13: PCR amplified product P0451 + J23101 colonies 1, 5 and 23 respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations P0451 with and promoter J23101..Lane1:Ladder. Lane 11,12 and 13: PCR amplified product P0451 + J23101 colonies 1, 5 and 23 respectively. The other lanes are samples from other experiments.

LuxY + J23101 ligation from colonies 1, 7 and 21.

Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lane 6,7 and 8: PCR amplified product LuxY (Mr.gene) + J23101 colonies 1, 7 and 21 respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lane 6,7 and 8: PCR amplified product LuxY (Mr.gene) + J23101 colonies 1, 7 and 21 respectively. The other lanes are samples from other experiments.

LuxY + J23101 ligation from colonies 5,10,11 and 19.

Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lanes 12,13,14 and 15: PCR amplified product LuxY (Mr.gene) + J23101 colonies 5,10,11 and 19,respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lanes 12,13,14 and 15: PCR amplified product LuxY (Mr.gene) + J23101 colonies 5,10,11 and 19,respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lane 2: PCR amplified product LuxY (Mr.gene) + J23101 colony 24.The other lanes are samples from other experiments.
Colony PCRs:Ligations LuxY with promoter J23101..Lane1:Ladder. Lane 2: PCR amplified product LuxY (Mr.gene) + J23101 colony 24.The other lanes are samples from other experiments.

Lumazine + J23101 ligation from colonies 1,4 and 8.

Colony PCRs:Ligations Lumazine with promoter J23101..Lane1:Ladder. Lane 6,7 and 8: PCR amplified product Lumazine (Mr.gene) + J23101 colonies 1, 4 and 8,respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations Lumazine with promoter J23101..Lane1:Ladder. Lane 6,7 and 8: PCR amplified product Lumazine (Mr.gene) + J23101 colonies 1, 4 and 8,respectively. The other lanes are samples from other experiments.

Lumazine + J23101 ligation from colonies 2,3,5,6 and 7.

Colony PCRs:Ligations Lumazine with promoter J23101..Lane1:Ladder. Lanes 7,8,9,10 and 11: PCR amplified product Lumazine (Mr.gene) + J23101 colonies 2,3,5,6 and 7,respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations Lumazine with promoter J23101..Lane1:Ladder. Lanes 7,8,9,10 and 11: PCR amplified product Lumazine (Mr.gene) + J23101 colonies 2,3,5,6 and 7,respectively. The other lanes are samples from other experiments.

Lumazine from Mr.Gene + plamid pSB1C3.

Colony PCRs:Ligations Lumazine with plasmid pSB1C3..Lane1:Ladder. LaneS 5,6 and 7: PCR amplified product Lumazine (Mr.gene) in plasmid pSB1C3 colonies 1, 17 and 22,respectively. The other lanes are samples from other experiments.
Colony PCRs:Ligations Lumazine with plasmid pSB1C3..Lane1:Ladder. LaneS 5,6 and 7: PCR amplified product Lumazine (Mr.gene) in plasmid pSB1C3 colonies 1, 17 and 22,respectively. The other lanes are samples from other experiments.





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