RNA pellet resuspension in EtOH -- How??

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Sep 21 20:28:34 EST 2000


I'm not sure I fully understand your protocol! Ae you sure have the right
protocol? 

After you precipitate the RNA with LiCl (2-2.5M is standard), you have to
resuspend the LiCl-pptd RNA in either water or TE. (It is also a good idea
to wash the pellet once with 2M LiCl prior to this resuspending step.)
After the RNA has completely gone into solution, you will then re-ppt it
with etoh (2.5 volumes). This setp usually does not need any further
addition of salt. After the RNA has presipitated out in etoh (usually
several hours at -20 C if the conc is low) you will spin the RNA down and
then wash the pellet with 67 to 70% etoh. It's not a great idea to use any
higher conc of etoh. The function of this wash is to wash off as much of
the residual salt as possible, and etoh at more than 70% does a very poor
job of it. I have not come across any ref that claims to get rid of
macromolecules (carbohydrates, proteins, lipids, etc.) by 80% etoh wash.
RNA (or DNA) will NOT resuspend in 70% etoh, so don't even try it! You will
damage the integrity of the nucleic acid if you try that. 

Hiranya.


At 06:08 PM 9/21/00 GMT, ghenipus at my-deja.com wrote:
>Just a small techniques question:  I'm (learning how to) prepare total
>RNA from sorghum leaves for the purposes of Northern Analysis.  Though
>the exact citation eludes me for the moment, we're using a varient on
>hot phenol to extract (no guanidium thicynate, no BME, etc.)  I've done
>RNA extraction with one other protocol before, and I get the impression
>that an overnight LiCl incubation is pretty standard for selectively
>precipitating out RNA following Chloroform extractions, and followed by
>an EtOH wash (70 or 80%) to get rid of some of the co-precipitating
>carbs.  My problem is this; in order to guarentee that all of the carbs
>dissolve in the EtOH and are hence eliminated, that pellet of RNA coming
>out of LiCl has to be resuspended pretty darned well in EtOH for the
>washes (we do two of them).  But that pellet absolutely doesn't want to
>resuspend unless I brutalize it, and then I run the risk of breaking
>up the transcript that I'm trying to recover here.  I'm under the
>impression that anything more vigorous than a brisk pipetting up and
>down is too much (i.e. no vortexing, no shaking it, or flicking it, or
>mixing it with the tip, or rubbing it up and down a plastic block), but
>pipetting up and down isn't doing much to break up these pellets.  Is
>there some little trade secret that I'm not privy to for getting RNA
>pellets to resuspend that's more time-efficient and easier on
>my thumb?
>
>Thanks,
>
>Adam
>
>
>Sent via Deja.com http://www.deja.com/
>Before you buy.
>
>
>
Dr. Hiranya Sankar Roychowdhury
Dept. of Molecular Biology	
PO Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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