RNA resuspension problem
njuni at poppy.ocn.ne.jp
Thu Jun 21 06:39:23 EST 2001
The gel-like substance may be polysaccharides.
The same thing sometimes happens to me when the amount of starting material
is too much as against the volume of RNA extraction system.
2 possible solutions to remove polysaccharides from the pellet:
1) Second round of RNA purification from the pellet: For example, add TRIZOL
to the pellet (or suspension), and proceed to homogenization and extraction.
2) LiCl (w/o alcohol) precipitation: Resuspend the pellet in 2M ("Molecular
Cloning" says 0.8M) LiCl with pestle or pipetting, and centrifuge at 4C.
Large RNAs (mRNAs) precipitate, while polysaccharides and small RNAs (tRNAs)
in article 20010619150239.15314.qmail at ww02.jatek.com, Melanie at
mmhierl at yahoo.com wrote on 6/19/01 3:02 PM:
> I have in the past had no problems precipitationg and resuspending my RNA in
> DEPC H20. However, the last time I tried to do this using 1/10 volume NaAc
> ph 5.5 and 2 volumes EtOH and freezing at -20 overnight, I come up with a
> clear gel like blob of a pellet that I can't get to redissolve. I have
> heated at 65 for 10 min several times, vortexed, flicked the tubes
> repeatedly, etc. Should I heat the tubes longer? Will that harm the RNA?
> Any other suggestions?
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