RNA resuspension problem (heat issues)
alansmith at students.wisc.edu
Tue Jun 19 17:33:57 EST 2001
I have a quick question about this remark.
>Heating for long period is not good.
I always heat my samples for 5-20 minutes at 65 C after a RNA prep to help
dissolve the RNA in formamide. I have not noticed damage often, but some
times I loose a sample. Does high heat cause the RNA to autocatalyse itself
and degrade? What is the general reason not considering RNases that would
cause heat to destroy a sample of RNA in solution (assuming no RNases)? I
know that metal ions can cause RNA to degrade itself and freeze thawing can
cause a large problem, but I have always assumed that RNA is stable at high
temps 60 - 95 C. Does the heat just speed up the weird autocatalytic
activity of RNA and cause a sample to degrade?
From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]On Behalf Of "SURESH KUMAR K.G."
Sent: Tuesday, June 19, 2001 2:45 PM
To: methods at hgmp.mrc.ac.uk
Subject: Re: RNA resuspension problem
This could be due to polysaacharides getting into your RNA prep. May be
you are going a bit too close to the interphase while pipetting out the
sup after phenol:chloroform extraction. Try avoiding this..your
RNA will be fine.
Heating for long period is not good.
One more thing you can check...just spin down the gel like pellet..and
check out the sup for RNA.
Hope this helps.
On 19 Jun 2001, Melanie wrote:
> I have in the past had no problems precipitationg and resuspending my RNA
> DEPC H20. However, the last time I tried to do this using 1/10 volume
> ph 5.5 and 2 volumes EtOH and freezing at -20 overnight, I come up with a
> clear gel like blob of a pellet that I can't get to redissolve. I have
> heated at 65 for 10 min several times, vortexed, flicked the tubes
> repeatedly, etc. Should I heat the tubes longer? Will that harm the RNA?
> Any other suggestions?
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