IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/17

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Ligation Procedure:Lumazine/LuxY with plasmid pSB1C3 and promoters J23101/J23102.

I'm working in Step 7 of the Ligation Procedure: Lumazine/LuxY with plasmid pSB1C3 and promoters J23101/J23102.

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

Results:Ligations Lumazine/LuxY with plasmid pSB1C3 and promoters J23101/J23102.Procedure Step 7

After re-culturing the colonies transformed with each ligation mixture, only colonies harboring the Lumazine/LuxY Mr.gene constructions fused to J23101 and J23102 grew correctly.

  • Colonies selected to do the colony PCR reactions:

Lumazine + J23101 ligation from colonies 1,5 and 15.

Lumazine + J23102 ligation from colony 1,6 and 12.

LuxY + J23101 ligation from colony 1, 2 and 15.

LuxY + J23102 ligation from colony 2,4 and 5.

Colony PCRs Ligations Lumazine/LuxY+promoters J23101 and J23102..Lane1:Ladder. Lane 2,3 and 4: PCR amplified product LuxY + J23101 colonies 1, 2 and 15 respectively.Lane 5,6 and 7:PCR amplified product LuxY + J23102 colonies 2,4 and 5, respectively. Lane 8,9 and 10:PCR amplified product Lumazine + J23101 colonies 1, 5 and 15, respectively. Lane 11,12 and 13:PCR amplified product Lumazine + J23102 colonies 1, 6 and 12, respectively.
Colony PCRs Ligations Lumazine/LuxY+promoters J23101 and J23102..Lane1:Ladder. Lane 2,3 and 4: PCR amplified product LuxY + J23101 colonies 1, 2 and 15 respectively.Lane 5,6 and 7:PCR amplified product LuxY + J23102 colonies 2,4 and 5, respectively. Lane 8,9 and 10:PCR amplified product Lumazine + J23101 colonies 1, 5 and 15, respectively. Lane 11,12 and 13:PCR amplified product Lumazine + J23102 colonies 1, 6 and 12, respectively.



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