dissolving of RNA

David F. Spencer dspencer at is.dal.ca
Mon Mar 24 16:11:10 EST 1997


In article <33314AD5.475F at mpiz-koeln.mpg.de>, maiwald at mpiz-koeln.mpg.de wrote:

> Hi, all,
> 
> I have problems to dissolve RNA-pellets in DEPC-treated H2O. I deal with
> barley total RNA that was extracted with phenol and precipitated with
> acetic acid/ ethanol and with LiCl. Although I keep the samples at 65 C
> and increased the volume several times, the pellets do not dissolve (or
> very, very, very slowly).
> 
> Does anybody have any tips how to overcome this problem?
> 
> Thanks                                  Frank

This seems to be a recurring theme in this newsgroup.

First I'll assume that the pellet you've purified is largely (or entirely) RNA,
and not heavily contaminated with carbohydrate, otherwise it's a different
problem.

After you precipitate the 'large' RNAs with 1M LiCl (or 1M or 3M NaCl) you must
wash the RNA precipitate with 70-80% ethanol to remove all traces of salt
(and be sure that all of the inside surfaces of the tube get washed).  This
step can be difficult if the pellet is large and tightly packed. If you are
salt fractionating relatively large quantities of RNA, be conservative in how
long and fast you spin down the precipitate so as to make the washing steps
easier.  Ideally you want to resuspend the RNA pellet as thoroughly as possible
to eliminate the residual salt. You may have to give the RNA more than one wash.
If you really need to eliminate the small, salt soluble RNAs, do the minimum
spin to get the RNA lightly pelleted and then wash the RNA with the 1M LiCl (or
NaCl) and respin. Of course if you can't tolerate any sRNAs then you'll have to 
repeat the salt fractionation anyway.

If you don't eliminate salt the RNA will be next to impossible to redissolve
because the small volumes of water (or TE) used can cause the salt concentration
to get to 1M or higher; obviously this is how you precipitated the stuff in the
first place so it's not going to redissolve. [You can always rescue the RNA if
you get in this mess by adding a small amount of NaOAc or NaCl solution, maybe
a 20th volume of 3M say, then add 2-3 volumes of 95-100% ethanol and treat 
it like a standard etol ppt'n; you won't lose any RNA that did redissolve 
because it will reprecipitate and you will get rid of some or all of the 
original carryover salt because it probably redissolved easily.]

As to this (apparently popular) notion that you can overdry an RNA pellet, let
me state that this is pure hogwash.  If RNA is properly desalted then you can
dry it for a week and it will redissolve just fine.  Note that this is quite 
different from the situation for larger quantities of even well washed high 
molecular weight (greater than 20-30 kb) _DNA_.  In this case it is definitely 
unwise to do more than a light drying if you wish to get the DNA
resolubilized in less than days or weeks.

Dave

-- 
David F. Spencer, PhD
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia, Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca



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