IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/23

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Working on LovTAP from Mr.Gene: Gel extraction & Ligation

Once LovTAP plasmid from colony 9 was digested with XbaI/PstI and EcoRI/PstI, the resultant fragments were used to extract LovTAP from the gel, using the previously described protocol.

Resuts: LovTAP Gel extraction

We extracted succesfully LovTAP from colony 9 digested with XbaI/PstI, and EcoRI/PstI.

According to the next image the LovTAP extraction of both restriction reactions from the gel band around 882nt was successful and there was not contamination from other bands.

Considering this result we decided to continue with the ligation procedure in order to join LovTAP with the previously choosen promoters. So that, I also loaded the plasmids harboring the promoters that were digested and dephosphated for ligation -Lane 2 to Lane 8- to calculate the quantity of the samples that must be used for ligation.

Gel extraction: LovTAP from Colony 9 digested. Lane1:Ladder. Lane2:J23102.Lane3:J23105.Lane4:J23106.Lane5:J23110.Lane6:J23114.Lane7:J23116.Lane8:J23117.Lane9: LovTAP colony 9 digested with XbaI/PstI. Lane10: LovTAP colony 9 digested with EcoRI/PstI.The other lanes are samples from other experiments

Working on CcaS and CcaR synthesized plasmid from Mr.Gene: Edinburgh Shipment

I am going to do a PCR reaction by duplicated in order to amplify the CcaS and CcaR construction synthesized. This PCR product will be send to Edinburgh team.

The product 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 CcaS and CcaR from Mr.Gene

  • Reaction 1
Reactive Quantity
MgCl2 3μL
Buffer 3.3X 6μL
dNTP's 5μL (0.4mM)
Primer Forward 3μL (5pmol/μL)
Primer Reverse 3μL (5pmol/μL)
DNA template (Mr.Gene Plasmid:CcaS-CcaR 2μL + 8μL 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 added to reaction 1.

Each cycle is programmed as follows:

Temperature Time
94°C 45s
60°C 45s
72°C 3:30 min

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

Results:PCR CcaS and CCaR

CcaS and CcaR construction was well amplified. The PCR product from each replica was around the expected size 3204 nt

CcaS&CcaR amplified product. Lane1:Ladder. Lane11: Ccas & CcaR replica 1. Lane12: Ccas & CcaR replica 2.The other lanes are samples from other experiments