Effect of Isopropanol on mRNA

Iain Shaw iainshaw at eircom.net
Fri Apr 27 05:34:31 EST 2001


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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