Effect of Isopropanol on mRNA

Alan Smith alansmith at students.wisc.edu
Fri Apr 27 09:42:04 EST 2001


The IPA probably is not drying and the residual contamination is causing a
problem with RT-PCR.  If the IPA is cleaned from the tube I don't see why it
would cause a problem.  I have been lazy and not washed RNA with 70%ETOH
after ppt with IPA and did not have a problem in RT-PCR. I used total RNA
though, but could mRNA be that much different? Could there be a problem with
program or primers?
Alan Smith

-----Original Message-----
From: owner-methods at hgmp.mrc.ac.uk
[mailto:owner-methods at hgmp.mrc.ac.uk]On Behalf Of Iain Shaw
Sent: Friday, April 27, 2001 5:35 AM
To: methods at hgmp.mrc.ac.uk
Subject: Effect of Isopropanol on mRNA


Mornin all,

May be barking up the wrong tree, or clutching at straws or other such
things here but I have a question for you all.

We have been carrying out some RT-PCR to construct recombinant antibodies
from mouse spleen.  Total RNA has been isolated, and then mRNA isolated
using Pharmacia spin-columns, finally first strand synthesei carried out
using the RT portion of the Pharmacia Recombinant antibody system kit.

This has been working very well and we have constructed several
antibodies.  Recently a student in the lab has been trying and always
fails on the RT-PCR step, with either no band or smears (conditions are
VERY well worked out, and the PCR machine has not changed).  Examining
the lab book of the student I noticed that at the end of the mRNA
isolation step wheras the protocol says to ppt the mRNA with glycogen,
Kacetate and EtOH at -20 and then spin and allow pellet to dry before
resuspension in DEPC-H20 the student had then washed the mRNA pellet in
100% isopropanol.  This appears to be the only difference in an otherwise
successful protocol, and to be quite honest I cannot work out what the
effect of the Isopropanol (if any) could be.  I would be more than happy
to receive suggestions on whether this would have had an effect or if I
am indeed grasping at straws.

Many thanks.

Iain



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