Help-Lg-scale Bacterial Genomic DNA Prep
Nick C.K. Heng
hengnck at sanger.otago.ac.nz
Thu Sep 5 00:37:28 EST 1996
In article <501np0$l2m at mule2.mindspring.com>
baderja at aub.mindspring.com (Joel Bader) writes:
> I'm seeking some advice on large-scale genomic DNA preps. I have a
> bacteria (gram-negative) that I want to pop and extract pure genomic
> DNA, but the organism produces tremendous amounts of polysaccarides,
> gycoproteins and other general protein contaminates. I have up to now
> been using several large scale chemical extraction methods, i.e.
> Marmur procedure and Cetyltrimethylammonium Bromide lysis with little
> success. Can someone help? I don't get any DNA in my last extraction
> steps and the DNA refuses to come out of solution during the proper
> ethonal extractions. I suspect that my original lysate was
> incompletely lysed or that the DNA stuck to the
> polysaccarides/proteins and was spun out with with them in an earlier
> phenal-CH3 extraction/centrifugation step. Thanks in advice for any
> help anyone wishes to provide.
> Joel Bader<jabader at mindspring.com>
Have you considered trying embedding your bacterial cells in agarose
plugs and then lysing them in situ? We had a problem with the bacterial
strains in our lab having contaminating material and/or nucleases.
Lysozyme followed by proteinase K treatment gave genomic DNA good
enough for restriction analysis, etc. The "pasteur pipette spooling"
method (is this the Marmur procedure?) didn't work.
You may not get tons of DNA with agarose plugs, though.
Just a thought...
Dept. of Microbiology
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