IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/19

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Working on LuxY synthesized plasmid from Mr.Gene

In order to test the correct lenght LuxY transformed, I am going to do a PCR reaction using as template the plasmid transformed harboring LuxY and the restriction fragments obtained with said plasmid.

Primers:

Forward:Preffix primer

Reverse:Suffix primer


PCR: Plasmid transformed harboring LuxY

Reactive Quantity
MgCl2 2.5μL
Buffer 10X Taq 5μL
dNTP's 2.5μL (0.4mM)
Primer Forward 2.5μL (5pmol/μL)
Primer Reverse 2.5μL (5pmol/μL)
DNA template (Plasmid LuxY transformed 2μL + 8μL HPLC) 4μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture


PCR: Fragments obtained from restrictions of Plasmid LuxY transformed


Reactive Quantity
MgCl2 2.5μL
Buffer 10X Taq 5μL
dNTP's 2.5μL (0.4mM)
Primer Forward 2.5μL (5pmol/μL)
Primer Reverse 2.5μL (5pmol/μL)
DNA template (Restriction fragments from Plasmid LuxY transformed) 1μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture

Results LuxY PCRs reactions:

The product obtained from the two PCR reactions was around 2000 nt of size. Considering that the correct size is 820 nt, we are going to repeat the transformation of the plasmid, it's possible that we did a mistake and chosen the wrong plasmid.

I'm going to do another PCR reaction using as template the plasmid from Mr. Gene that harbors LuxY.