User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/06/17

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χρόνος πέρασμα June 17th 2011


  • pBBRMCS5 digestion with ApaI. Test to see if the bacteria host for this plasmid is DCM+.

  • Today I made the pBBRMCS5 plasmid digestion again. The previous digestion couldn't be used to see from which bacteria host come the plasmids as we mistakenly put to digest something that didn't even possess ApaI sites. The point of this digestion is the following: As E. coli DH5α has activated its methylase DCM, the sites that are recognized by the restriction enzymes ApaI and SacI must come methylated. These enzymes cannot recognize their proper sites if the DNA is methylated. As our new assembly design involves the use of these enzymes, we must obtain our plasmids from a source that does not methylate them. That's way we want to digest with only one of the restriction enzymes, just to test if the plasmids come from DH5α E.coli.
  • The digestion was performed at 25°C with the followings reactants:
2.5 ml Tube
6 µl H20
1 µl Buffer #4
2 µl DNA (pBBRMCS5)
1 µl ApaI Enzyme
10 µl Total
  • The digestion was left overnight. If the electrophoresis gel shows up to have the same pattern as the non-digested plasmid, then, pBBRMCS5 indeed comes from DCM+ bacteria. We should transform this plasmid to other E.coli strain that is DCM- , E.coli BL21(DE3) is a good candidate.