RNA pellet resuspension in EtOH -- How??

ghenipus at my-deja.com ghenipus at my-deja.com
Thu Sep 21 13:08:18 EST 2000


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


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