User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/02

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Plasmid Purification

ABSTRACT

  • Plasmid purification using the "High Pure Plasmid Isolation Kit" of Roche in order to test the reliability of the kit.

  • Today´s goal was isolate plasmid DNA of E. coli. The desired plasmid is called pBBRMCS5. For this purpose we use the "High Pure Plasmid Isolation Kit" of Roche. We follow the steps indicated in the Instruction manual, pages 8 and 9. You can see this steps in the following link:

Instruction Manual

Because the E. coli strain used doesn´t have high nuclease content we did not perform the optional wash step.

After obtaining the samples, we did an electrophoresis with a 1% agarose gel, 80 V x 100 min. The proportions of the samples were 3μL of dye and 5 μL of plasmid DNA. Unfortunately the gel showed no visible bands in the lanes of plasmid DNA.

We did another electrophoresis using again a 1% agarose gel, 125 V X 30 min, with other sample proportions: in one lane 3μL of dye and 7μL of plasmid DNA, in a second lane 3μL of dye and 10μL of plasmid DNA. This new gel showed two bands, but not very clear that correspond to chromosome DNA. You can look for the picture in Pablo´s notebook.

The conclusion of this experiments is that something wrong is going on with the plasmid extraction kit. On monday we will try to find out what is wrong.



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