IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/18

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Working on CCaS/CCaR and LuxY synthesized plasmid from Mr.Gene

In order to test the correct lenght for CCaS/CCaR and LuxY synthesized, I am going to do a PCR reaction using the following primers:

Forward:Preffix primer Reverse:Suffix primer

After the PCR we are going to do a restriction enzyme assay to test the products.


PCR: Plasmid transformed harboring CCaS/CCaR

Reactive Quantity
MgCl2 2.5μL
Buffer 10X Taq 5μL
dNTP's 5μL (0.4mM)
Primer Forward 3μL (5pmol/μL)
Primer Reverse 3μL (5pmol/μL)
DNA template (Plasmid CCas/CCaR 2μL + 8μL HPLC) 2μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture


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) 10μL
Taq polymerase 1μL
HPLC Up to a final volume of 50 μL of the mixture

PCRs Results

According with the next image CcaS PCR reaction yields a product around the expected size 3204nt however the fragment amplified in the LuxY PCR reaction has a size around 2000 nt, thus not corresponding to the expected size that is 820 nt.

I’m going to repeat the PCR reaction with a shorter Elongation step.

PCR CcaS and LuxY synthesized. Lane1:Ladder. Lane4: CcaS 3. Lane5: LuxY. The other lanes are samples from other experiments
PCR CcaS and LuxY synthesized. Lane1:Ladder. Lane4: CcaS 3. Lane5: LuxY. The other lanes are samples from other experiments

Working on LovTAP synthesized plasmid from Mr. Gene

LovTAP extraction from gel

In order to extract LovTAP from the gel ready for ligation with J23 promoters and plasmid pSB1C3 , I'm going to do two replicates of enzyme restriction reactions using XbaI and PstI to cut LovTAP plasmid isolated from colony 1, as well I will cut the same plasmid using EcoRI and PstI.

LovTAP colony 1.Restriction enzyme assay:Preparation for Ligation with J23 promoters and plasmid pSB1C3

  • Ligation for promoters: Restriction enzymes XbaI and PstI.
Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture


  • Ligation for plasmid pSB1C3: Restriction enzymes EcoRI and PstI.
Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture

Results:LovTAP colony 1.Preparation for Ligation with J23 promoters and plasmid pSB1C3

The plasmid of LovTAP from colony 1 was successfully digested in both restriction enzyme reactions.




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