Do NTP's precipitate with ethanol? How efficiently?
Dave Hollingworth
do they both go into the pellet? Do the conditions for precipitation
matter (different salts, temperatures etc....). Is there a better way
of precipitating NTP's?
Dave
CK Wen
I do not think dNTPs precipitate with the DNA, though no direct
evidence can be provided to you. Just think that when we prepare
P32-end-labeled DNA probes, the gamma-P32-ATP is removed after ethanol
precipitation. Also, if you EtOH ppt your alpha-P32-lableled DNA
probes, you may get "hot" sup which indicates that the free dCTP is in
the EtOH phase. After several EtOH washes, the sup is never hot and the
pellet should be the labeled DNA.
Hope this help.
Cheer,
Chi-Kuang
Robert Slany
from my personal experience I can't agree with your statement. Although I do
believe that the efficiency is very, very low in an experiment done by myself
not only enough dNTPs but also Klenow enzyme survived ethanol precipitation
to fill in the ends of the following restriction digest.
The morale: don't save time by omitting heat inactivation. It took me a week
to figure out that the cloning was actually blunt ended!
Also in vitro transcribed and subsequently precipitated RNA contains tons of
nucleotides that have to be removed by chromatographic steps.
If you need it absolutely nucleotide free, do a DEAE-Sepharose purification.
Just my 0.02$
Robert
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Anton Scott Goustin
Deoxyribonucleotide triphosphates are quite insoluble in mixtures of
EtOH and NaCl. For example, if you precipitate a randon-primer reaction
by first raising the NaCl to 0.3 M and then adding 2.5 volumes of EtOH,
a substantial fraction of your dNTPs will come down with the DNA. The
solubility of dNTPs, however, is substantially reduced in ammonium
salts. For example, a clean separation between dNTPs and DNA can be
achieved by first raising the concentration of ammonium chloride to 4
molar and then adding 2.5 vol EtOH.
Ppttn of dNTPs also depends on the base. G is most insoluble, A is
most soluble. The ribo versions of each NTP are also more soluble than
the deoxy versions.
David Martin
Anton Scott Goustin <a...@cmb.biosci.wayne.edu> wrote:
-->The information in Chi-Kuang's reply (above) is quite misleading.
-->
-->Deoxyribonucleotide triphosphates are quite insoluble in mixtures of
-->EtOH and NaCl. For example, if you precipitate a randon-primer reaction
-->by first raising the NaCl to 0.3 M and then adding 2.5 volumes of EtOH,
-->a substantial fraction of your dNTPs will come down with the DNA. The
-->solubility of dNTPs, however, is substantially reduced in ammonium
-->salts. For example, a clean separation between dNTPs and DNA can be
-->achieved by first raising the concentration of ammonium chloride to 4
-->molar and then adding 2.5 vol EtOH.
How as you are precipitating both? This does not make any sense. Either that
or there is a step or two missing
.d
-->
-->Ppttn of dNTPs also depends on the base. G is most insoluble, A is
-->most soluble. The ribo versions of each NTP are also more soluble than
-->the deoxy versions.
-->
-->
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