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.



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


 * Reaction 2


 * 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:

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




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