Clostridial genomic DNA

billing at vetsci.microvet.arizona.edu billing at vetsci.microvet.arizona.edu
Fri Jun 13 17:31:22 EST 1997


Does anyone have any thoughts on how to prepare genomic DNA from species
of Clostridium other than C. perfringens?

I am trying to prepare genomic DNA from C. novyi type B and C.
haemolyticum (or novyi type D).  I have no trouble making C. perfringens
DNA and I can get the other strains to lyse without trouble.  But the
yields of DNA are low and rapidly degrade (e.g., O/N at 4oC or as soon
as I try to cut them).

I am guessing this is due to the action of endogenous DNAses from the
clostridia, but how do I get around it?  I have tried using
SDS/proteinase K lysis followed by various combinations of phenol and
CHCL3 extraction.  I have also tried using a Qiagen genomic prep kit
which uses guanidine in the solubilization buffer.  This works better
(ie., the DNA doesn't degrade as quickly), but the yields are usually
10-20 fold less than expected.

Any thoughts or suggestions would be appreciated.

HJ



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