CTAB method for genomic DNA isolation from Salmonella

Rafa Maldonado Rafael at genetics.med.utah.edu
Thu Jan 5 21:56:15 EST 1995


In article <RANKD.1.0008BF44 at FOODSCI.PURDUE.EDU>
RANKD at FOODSCI.PURDUE.EDU (Doug Rank) writes:


> but have been getting precipitates after both the NaCl addition step as well 
> as after the CTAB/heating step.  Looking at the centrifuged precipitates 

Hi Doug:
I have been using the CTAB method for years in different bacteria
species. There is a critic point when adding the NaCl (and the CTAB):
you have to get mixed very well before the next step. When adding the
NaCl, the cell suspension is a lysate very viscous, and is very
difficult get the drop of NaCl dissolved. I usually vortex 30 s. or
more, and the solution get turbid, of course, but it must to be
homogeneous. If the NaCl and lysate solutions are not mixed properly,
you will get your DNA precipitated, as I think it is happening to you.
If still having problems with the mixing of NaCl, try heating at 65C
ten minutes and vortex again.
Anyway. I never looked for DNA in the precipitate nor in the
interphase. Maybe the DNA is going to the pot massively, but if you
finally get 2-4 ug per prep, it's still OK and I wouldn't care.
The big amount of RNA is normal: you are not doing a DNA extraction in
the strict sense, but a total nucleic acid extraction. The RNA is
mainly ribosomal RNA. Get rid off it with RNAse A.

I hope this helps

Rafa

----------------------------------
Rafael Maldonado
University of Utah
Rafael at genetics.med.utah.edu



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