Re: tRNA cleanup/precipitation?

[Dmitry Bochkariov]

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
> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods

[Nick Theodorakis]

On Friday, November 8, 2013 11:59:39 PM UTC-5, 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!
>
>

Most silica-based binding columns do not perform well on small RNAs such as tRNA, at least not without modification. Several vendors sell columns for micro-RNA purification, which should work, although it might be overkill, expense-wise, since those kits will also include reagents you won't need for a simple clean up. Note that the trick to getting small RNAs to bind is a (most likely proprietary) additive added to the binding buffer, not the columns itself.

As indicated, ordinary sodium acetate/ethanol works just fine to precipitate tRNA; in fact, in some procedures, tRNA is often used as a carrier for precipitation.

Nick

[Christian Praetorius]

d...@no.email.thankstospam.net (DK) wrote:

>is the best way of cleaning up such reactions to remove salts and
>nucleotides? Without losing the precious RNA, of course.

I would go for ammonium acetate to remove the free nucleotides. tRNA
are used as a carrier for small amounts of DNA, so I wouldn't worry
too much. There is a interesting paper about it from the Bethesda
Research Laboratories: Crouse J, Amorese D (1987). "Ethanol
Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate".
Focus 9 (2): 3-5. The PDF can be found here:
http://bio.wayne.edu/profhtml/Cunningham/private/privatedocuments/EtOH.pdf
(and probably also somewhere on the Life Technologies website).

>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

There is another interesting paper also from BRL, which finds
surprisingly little effect of cooling in the precipitation at all:
Zeugin JA, Hartley JL (1985). "Ethanol Precipitation of DNA". Focus 7
(4): 1-2.
http://www.lifetechnologies.com/content/dam/LifeTech/migration/en/filelibrary/pdf/focus.par.56415.file.dat/focus%20volume%207%20issue%204.pdf

Christian

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