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Rehydration of DNA

Warren watchcity at my-deja.com
Tue Jan 18 09:54:22 EST 2000


Hello,	I presume that the DNA in question is genomic, not cosmid or smaller.
 I have only used the spool-DNA-onto-glass-rod method of recovery for cleaned
genomic DNA.  This method also requires an extended period for the DNA to be
solubilized.  1 suggestion would be to limit the drying time (at RT) after
the organic wash until the pellet looks dull glassy and there is no (easily)
discernable smell of alcohol.  I have resuspended at 37C, but only for
something like cosmid/plasmid maxipreps (in TE 10:1, pH~8). I always guessed
that the reason for not working well in PCR might have as much to do with
allowing the organic molecules to be released from the	(incredibly chaotic)
matrix of the precipitated (and I think denatured) DNA as anything else.  The
DNA molecules very likely have some residual protein included, and that could
act like mortar till an aqueous environment could be reestablished.  Think
salt encrusted wildly tangled stuck together super length strings, and the
time required to reach an equilibrium of greatest stable matching ds
coordination (since you wouldn't be assisted by the circular nature of the
vectors).  Perhaps there may be a concentration dependant effect where such
large molecules simply take a long time to reach a low enough dilution to
"allow" the PCR replication enzymes "in".

Since genomic is stored at 4C anyway, and gentle handling is crucial to
"optimizing" yield, why not use this step as a planned stopping point?	Plan
your overnight	PCRs for the following day from today's genomic preps.	When
in doubt, do both....  Warren

In article <388411aa.23358787 at news.tas.gov.au>,
  jeremy.carson at dpiwe.tas.gov.au (JCarson) wrote:
> We have noted that DNA eluted from silica with 10mM pH8 TRIS buffer
> does not amplify well by PCR if the  DNA is used immediately after
> elution. If the eluted DNA is stored at 4C overnight and used in a PCR
> then good amplification occurs.
> We presume that the poor amplification that occurs immediately after
> elution is because the DNA is not fully rehydrated.
> Any comments please about what is the nature of DNA rehydration. Why
> would a PCR not work well if the DNA is not adequately rehydrated? Is
> it likely that genomic DNA with high (or low) G+C content may
> rehydrate faster (or slower). Any suggestions as to how to speed up
> rehydration apart from raising the pH or warming the elution buffer.
> Any comments observations?
> Thanks
>
> Jeremy Carson
> Dept Primary Industry, Tasmania
> Australia
> jeremy.carson at dpiwe.tas.gov.au
>


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