User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/06/11

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Friday 11th June 2010

We are not sure if the plasmid containing the cI inverter actually has it, or if the part is consistent because of the lack of results with the restriction, that why I performed a PCR using the primers for prefix and suffix to extract the cI inverter from the plasmid, I made 3 tubes, one with the cI inverter containing plasid, and the negative and positive controls.

  • PCR with Taq Polymerase (30ul):


H2O...........40.5 ul
Buffer.........18 ul
dNTP’s.........9 ul
Mg(Cl)2........6 ul
Forward.......6 ul
Reverse.......6 ul
DNA............- ul
Taq Pol......1.5 ul

I checked both the new double restriction and the PCR in an agarose gel:


1º lane is the DNA ladder.
2º lane is the double restriction.
3º lane is the cI inverter’s PCR.
4º lane is the positive control.
5º lane is the negative control.


As it is clear in the image, the double restriction doesn’t shot the 1 kb band it should, only it shows the plasmid, the PCR came out very well.



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