DNA degradation

Paul R. Rohde P.Rohde at mailbox.uq.edu.au
Wed Oct 15 20:07:01 EST 1997


mirher at utu.fi writes: > Hello!
> Does anyone know how to inhibit DNase activity? I keep genomic DNA 
> samples at 4 C, and after 2 weeks the DNA seemes to degrade so that
> it can't be used for PCR etc...has anyone got same kind of problems,
> i would really need some ideas here! thanks a lot,
> 
> mirkka


Mirkka,

First of all is your gDNA clean?  I don't know how
you have extracted it but maybe you could do a
phenol:chlorform + chloroform extraction and see
if that makes a difference.  Try digestion with 
protease before this as well.
Is your TE autoclaved?  Pippetters clean?  Do you
fill your tip boxes with gloves, do you breath over
your samples?  DNA is robust but something is going
on.

Now for two suggestions which I haven't tried myself,
they are just suggestions.

A)  You could store the DNA in 50% glycerol (just add
equal volume glycerol to new DNA)and keep
it at -20 C.  Enzymes are stored in glycerol so they
won't freeze, so when adding your DNA to reactions
the glycerol should not be a problem.  If doing
restriction digests check with the NEB catalogue
about star activity for your enzyme.  Excess
glycerol can cause star activity (non specific
cutting).

B).  RNA is commonly stored in formamide to 
safely store it at 4, -20 or -70.  I've read
that the RNA can be put into RT reactions straight
if stored in formamide.  So again the formamide
shouldn't inhibit further reactions (??)
 If it preserves RNA it will preserve DNA!

RNA Ref:
TI: 	Solubilization in formamide protects RNA from degradation.
AU: 	Chomczynski-P
SO: 	Nucleic-Acids-Res. 1992 Jul 25; 20(14): 3791-2

I've read also that the formamide will need to be deionized
using a mixed bed resin, or buy it deionized.  The formamide
will last up to six months, then it could be tossed.
If it smells like fish/ammonia it can break down RNA.


If you try one of my siggestions and it works, let me know!

Best wishes,


Paul
Surgery, UQ.
Australia.




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