rna 260/280 ratio

Jim Hutchins hutchins at fiona.umsmed.edu
Fri Aug 25 07:31:31 EST 1995


Mrs R M D Lastrucci (075rlast at chiron.wits.ac.za) wrote:

: Dear All

: I am having problems with RNA extractions: 
[stuff deleted]
: spec. reading at A=260nm and A=280nm.

: Thanks in advance for any help and suggestions

It's a good procedure to also check the A230 and A325.  According to Ausubel
et al. (_Current Protocols in Molecular Biology_, "The Big Red Book"), A230
values greater than the A260 indicate organic solvent contamination, while
A325 values other than zero (or much, much less than the A260, if it is
very high) indicate particulate contamination.

For example, we find that isopropanol absorbs strongly at A230.  Any trace
of alcohol (ethanol or isopropanol) will inhibit RT, though I don't have
a reference for that handy.

Another possibility is that the RNA is completely degraded, but I would
guess that you have some alcohol in the final prep.  It's hard to get the
pellet dry enough to remove the alcohol, but still not bone-dry so that
it will not go back into solution.  One way is to heat the sample to 65
deg C after adding back the water to the dried pellet.

Traces of phenol or guanidium also give high A230 values.  Leftover phenol
I can usually smell.

There are other possibilities, but I think this is the most likely.

Hope this helps...
--
Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/    ***    E-mail:   hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68



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