IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/24

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Working on LovTAP and LuxY synthesized plasmids from Mr.Gene: Edinburgh Shipment

I am going to do PCR reactions in order to amplify the LovTAP and LuxY construction synthesized. These products will be send to Edinburgh team.

The products obtained will be purified using the High Pure PCR Product Purification kit from Roche.


Primers:

Forward:Preffix primer

Reverse:Suffix primer


PCR template:

Plasmid harboring LovTAP and luxY from Mr.Gene

  • Reaction 1
Reactive Quantity
MgCl2 3μL
Buffer 3.3X 6μL
dNTP's 3μL (0.4mM)
Primer Forward 2.5μL (5pmol/μL)
Primer Reverse 2.5μL (5pmol/μL)
DNA template(Mr.Gene Plasmid LuxY 2μL + 8μL HPLC)OR(Mr.Gene Plasmid LovTAP 1μL + 9μL HPLC) 1μL
HPLC Up to a final volume of 30 μL of the mixture


  • Reaction 2
Reactive Quantity
Buffer 3.3X 9μL
RTTH enzyme 0.5μL
HPLC Up to a final volume of 20 μL of the mixture


  • 35 PCR programmed cycles:

The reaction starts at 95°C during 5 min. After this step, the reaction 2 is loaded to the reaction 1 sample.

Each cycle is programmed as follows:

Temperature Time
94°C 45s
60°C 45s
72°C 1 min

After the cycle 35, the temperature changes to 72°C during 10 min and ends at 4°C.


Results:PCR reactions LovTAP and LuxY

Four PCR reactions were done for LovTAP and LuxY. The resultant amplified products are showed in the next image. From lane 8 to lane 11, the products are of the LuxY PCR reactions, the strongest band correspond to LuxY with size 820nt. As there were other products, I am going to extract LuxY from gel, as was done previously for LovTAP.

In LovTAP PCR reactions, the amplified products were around the expected size -889 nt- that is the lenght of LovTAP synthesized. These PCR reactions will be purified using the High Pure PCR Product Purification kit from Roche.

PCR:LovTAP and LuxY.Lane1:Ladder.Lane8:LuxY.Lane9:LuxY.Lane10:LuxY.Lane11:LuxY. Lane12: LovTAP.Lane13:LovTAP.Lane14:LovTAP.Lane15:LovTAP. The other lanes are samples from other experiments
PCR:LovTAP and LuxY.Lane1:Ladder.Lane8:LuxY.Lane9:LuxY.Lane10:LuxY.Lane11:LuxY. Lane12: LovTAP.Lane13:LovTAP.Lane14:LovTAP.Lane15:LovTAP. The other lanes are samples from other experiments



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