RNA ppt for Northerns

Eric C. Anderson e-anderson at ski.mskcc.org
Sun Oct 8 17:30:17 EST 1995


In article <458jr9$mll at mserv1.dl.ac.uk>, Denni Schnapp
<ds4 at st-andrews.ac.uk> wrote:

> Dear Bionet Folk,
Denni,

let's take these things one at a time...

1.  EtOH precipitation with NaOAc as you described works very well.  the
temperature you perform the precipitation at and the length of time you
let it go are really a personal preference.  i always do about 1 hour at
-20oC.  i know people who let it go for 10 minutes at RT and others who go
overnight at -70.  pick one that works for you and stick with it.  you can
spin at RT and you should do it for 15-20 minutes.

yes, you need to wash at least once with 70% EtOH and the best way to
ensure that it's RNAse free is to use a brand new bottle of 95-100% EtOH
and DEPC-water and make it  up in either RNAse free glassware or by
pouring out 30% of the EtOH and adding water to the original volume in the
EtOH bottle.

2.  if you can't DEPC treat something (like Tris buffers and pipet tips)
then i suggest autoclaving for 1 hour.  i would also like to take this
time to point out a comparison that a former colleague and i did once.  we
did 3 side-by-side northerns using identical rna and probes and the
following techniques:
   a.  totally anal-retentive DEPC treating, baking and autoclaving of all
materials.  new pipet tips on every dip (even in water going first into
tubes).  gloves (this is quite honestly the best thing you can do to
prevent RNA degradation).
   b.  just ordinary autoclaving and general good lab practices and
handling of all materials...pretend that you're doing a restriction digest
except wear gloves.
   c.  no sterilization, no gloves, etc.

the results were that "a" and "b" gave identical blots when it was all
over.  no degradation, good signal, etc.  as you may expect, "c" gave a
really nasty, streaky, degraded blot, but if you're not even going to
autoclave your pipets and wear gloves, then this is the sort of thing you
deserve.

3.> Finally, after running a formaldehyde gel the formaldehyde has to be removed
> prior to blotting which presumably leaved the RNA once again vulnerable to
> RNAses (or does it? I do not know anything about nucleic acids...). The
filter-
> paper used for capillary flow is in direct contact with the gel. This
> looks likea source of contamination, or is there a way of treating
> filterpaper?

no, you don't have to remove the formaldehyde before transfering.  you
only have to wash the gel into your transfer buffer (10X SSC in most
cases) and then transfer.  as for the filter paper, if it's contaminated
in the first place, then the RNA will degrade as it transfers.  any
company that would sell a filter paper for nucleic acid blotting that is
contaminated with RNAse should and would be laughed out of the
marketplace.  this is not to say that you will never have a membrane
problem or that a bad batch or 2 never occurs.  but more often than not
your membrane won't be a problem.  just keep it in the original package
and do not ever touch the box without gloves on.

good luck,

eric

-- 
Eric C. Anderson
Cell Biology and Genetics
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave., Box 470
New York, NY  10028
Phone: (212)639-2977
Fax:   (212)717-3298
e-anderson at ski.mskcc.org



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