Field of Science

Big prep of MAP7 DNA

We're almost out of the standard DNA that we use in our transformation assays.  It's chromosomal DNA of a strain called MAP7 (because it contains point mutations conferring resistance to seven different antibiotics).

So I grew up a liter of cells and prepped DNS from them.  Now I have 25 ml of nicely viscous DNA solution.  It's transparent and colourless but I suspect it's not really pure yet, so I'm doing a second purification on 0.5 ml just to check if the apparent concentration or purity changes when examined with the Nanodrop spectrophotometer.  I'll also do test transformations, with the last of the old DNA preps as a control.

Later:  The Nanodrop spec found that the repurified DNA had half the concentration of the big stock. So I ran a gel and found that the big stock still contains a lot of RNA.  My original RNase step must not have worked very well, probably because of the high concentration of SDS and of proteinase K.  So I';ve now incubated the prep with more RNase overnight.

1 comment:

  1. Hi Dr. Redfield,

    I''m from Dr. Brian Hammer's lab. I love your blog! I was wondering if there was a reference on the seven specific mutations that confer resistance to the markers you use.


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