DNA/RNA ethanol precipitation

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Thu Jun 1 16:15:11 EST 1995

In article <3qjv6t$ijt at aragorn.unibe.ch| vallan at ubeclu.unibe.ch writes:

| I have some quite basic questions concerning the centrifugation times
| for DNA and RNA precipitation with ethanol.
| Looking at different protocols I found suggestions for centrifugation
| times which range from 5 to 30 minutes in a microcentrifuge at full speed.
| Since loing I wonder what the optimal time really is. Is there more DNA
| recovery if one centrifugues 30 instead of 10 minutes? Is this dependend
| on the length and concentration of the DNA in the tube or on the volume
| of the DNA solution? What is the optimal centrifugation time for the
| washing step? (I found times from 2 to 15 minutes) And finally what is 
| the optimal incubation time at -70 degrees before centrifugation? Or is
| an incubation at -20 degrees enough?

All these questions concerning precipitation of DNA can be answered by reading
the article below. The optimal temperature, times, concentration, and volume of
DNA for 97% recovery are all spelled out for you. The best conditions are
actually 0-22 C, 10 minutes, 5ug/ml, and 20ul, respectively. Centrifuge for
15-30 minutes.

author = "J. Crouse
     and D. Amorese",
title = "Ethanol precipitation: ammonium acetate
as an alternative to sodium acetate",
journal = "B.R.L. Focus",
volume = "9",
number = "2",
pages = "3-5",
year = "1987"}

| While I'm at it: some people say that it is better if before putting
| the DNA in the cold bath one mixes the solution without vortexing it.
| Can somebody confirm this or is this just esoterics?

If your DNA is quite large, vortexing will cause shearing. Otherwise, it will
not hurt smaller plasmid DNAs. I always do pptn's by adding 1/5 th vol of 10 M
ammonium acetate, and 2.5 vol. of ethanol, invert, RT for 5 min., then spin 15
min....Done!  All that cooling in a water bath, etc. is unnecessary.

| I would very much appreciate if somebody could give me a reference
| where I could look up the answers to all these questions.

You're welcome

* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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