If you tRNA concentration is really >0.1mg/ml, it will precipitate with
EtOH just fine and virtually quantitatively.
Add Na salt to at least 100mM final and have pH neutral or slightly
acidic (pH 5.5 is good, and it is the best to use Na-Acetate buffer),
then add 2.5 volumes of EtOH chilled in a freezer. Mix and put your mix
in a freezer (-20C) for an hour or longer. Then spin in refrigerated
centrifuge. At this time it's OK for the mix to warm up to 0C or +4C.
tRNA precipitate will not dissolve.
You can repeat re-precipitation several times if you really need to
remove nucleotides completely. If you work carefully, you will not loose
much at all. Wash the pellet with 95% EtOH, but do it gently. Do not
vortex. All you do with the EtOH wash is remove residual Na-Ac.
If you do decide to do multiple re-precipitations, dissolve tRNA
precipitate in just water, then add Na-Ac again.
Overall, it is very easy to work with tRNA.
BTW, silica adsorption method so popular with DNA does not work well
with tRNA - yields are very poor.
Good luck
================================
Dmitry Bochkariov, Ph.D.
Principal Scientist
Advansta Inc.
1455 Adams Drive, Suite 1160
Menlo Park, CA 94025
(650) 325-1980 x530
================================
On 11/8/2013 8:59 PM, DK wrote:
This might be quite a silly question but just in case:
I will be making tRNA in vitro using T7RNAP. The in vitro reaction
contains crazy amounts of nucleotides (10 mM each NTP). What
is the best way of cleaning up such reactions to remove salts and
nucleotides? Without losing the precious RNA, of course.
Had it being DNA > 200 bp, I'd know what to do - either columns
or EtOH precipitation would do fine. I am unsure however what I
can expect from 85 bp tRNA.
For EtOH pption: How long to keep on ice? (Is low temp even
needed; for DNA I find that it is not beneficial at all). What cation
works best for small RNA? Also, some protocols suggest wash
with 95% EtOH to prevent loss. Good or not? The concentration is
expected to be high, > 0.1 mg/ml although the volume for now
is small, only 20 ul. Any benefit from adding linear polyacrylamide
under such conditions?
How do standard silica columns work for small RNAs? Anything needs
to be changed for binding? How does the capacity compare to
plasmid DNA?
Any other relevant advice is greatly appreciated. Thanks!
Dima
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