RNA pellet resuspension in EtOH -- How??

Savita Shah spshah at stanford.edu
Fri Sep 22 17:15:18 EST 2000


are you sure they are carbohydrates and not other junk (like proteins
etc..).
Also is your ppt. coloured? I think polyphenols may account for this
too. It is a good idea to use reducing agents like beta-mercaptoETOH
(1%), sodium metabisulfite (50-100mM, PVP or PVPP (1%)and/(or) thioUrea
(5mM) in the extraction buffer


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.






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