RNA ppt for Northerns

Denni Schnapp ds4 at st-andrews.ac.uk
Sun Oct 8 08:31:21 EST 1995

Dear Bionet Folk,

Trying to extract total RNA from crab hemocytes, I found I was left with
very low concentrations (ca 7 micrograms from 10^7 cells). I want to use
these samples for Northern blotting but I need to concentrate the RNA.
Presently the samples are dissolved in 50 and 80 microlitres of DEPC treated
water in microcentrifuge tubes. In Sambrook et al I have read that RNAs are
easily precipitated in 0.3M Sodium Acetate (best buffer to use prior to
formaldehyde gel electrophoresis) by adding 2.5-3 volumes ethanol and that
small volumes of sample can easily be precipitated in a single microcentri-
fuge tube, which is handy, but no further information is forthcoming from
that source. How long with the RNA/buffer/ethanol mix have to be incubated?
At what temperature (ice-bath, I presume...)? How long and how fast does it
have to be centrifuged (we have only unchilled microfuges and a chilled ge-
neral purpose centrifuge that pulls only ca. 4000g)? Does it have to be re-
incubated with ethanol like DNA seems to be? Any ideas?

Also, I try and keep my stuff RNAse-free in as much as possible but some of
the stuff is only treated by autoclaving. Is that insufficient to get rid
of RNAse? Is it recommended to increase the autoclave time from 15 to 30 mins
or something?

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?

Any advise would be welcome, I am a complete newcomer to these techniques!
Thanks everyone,


Denni Schnapp		    
Gatty Marine Laboratory    			Fax: (0)1334-463443
University of St. Andrews			e-mail: ds4 at st-and.ac.uk
St. Andrews Fife KY16 8LB   			
Scotland		   	http://www.st-and.ac.uk/~www_sa/personal/ds4/


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