coprecipitant for RNA

tfitzwater at NEXSTAR.COM tfitzwater at NEXSTAR.COM
Thu Mar 4 17:06:50 EST 1999


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0.5% Linear PolyAcrylamide (LPA) is a completely inert and cheap carrier used
for ethanol precipitations.   I modified the protocol from Gaillard, C. and
Strauss, F. (1990) Nucleic Acids Res. 18, 378 by eliminating the centrifugation
steps.
Prepare a 5% polyacrylamide solution in 40 mM Tris-HCl, pH 7.8, 20 mM sodium
acetate, 1 mM EDTA and polymerize with 0.1% APS and 1/1000 volume TEMED.  In a
50 mL Falcon 2098 centrifuge tube, mix in the order listed:  0.25 g acrylamide
(no bis-acrylamide) with 4.25 mL of Type I water, 200 
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=B5L of 1 M Tris-HCl, pH
8.0, 33 =B5L of 3 M sodium acetate, pH 7.5, 10 =B5L of 0.5 M EDTA and 5=
0 =B5L of 10%
APS.  Add 5 =B5L of TEMED and mix well to begin polymerization.  Incuba=
te at room
temperature for 30 minutes.  Add 12.5 mL (subscript:  )of 100% ethanol =
and mix
by inversion to cause immediate heavy precipitation.  3-5 minutes of ge=
ntle
inversion should gradually create a large white egg-shaped ball of prec=
ipitated
polyacrylamide.  Centrifugation is not required.  Pour off the supernat=
ant and
wash with 10 mL of room temperature 70% ethanol.  Mix by inversion and =
carefully
decant the ethanol.  Use a pipettor to remove the residual ethanol and =
let the
"pellet" air dry briefly.  Complete evaporation of the ethanol is not r=
equired,
as this solution is destined for ethanol precipitations.  Resuspend the=
 pellet
to the 50 mL mark of the Falcon tube with Type I water.  Go home and al=
low the
pellet to dissolve overnight.
Mix briefly and prepare 1 mL aliquots of 0.5% LPA and store at 4=B0C (s=
table over
1 year).  I have also successfully premixed this carrier with 7.5 M amm=
onium
acetate for elimination of one pipetting step.  The mixture has been st=
able for
1 year (so far) at room temperature.  RNA/DNA pellets containing LPA sh=
ould be
resuspended and incubated in a 37 degree heat block for 20 minutes.

Tim Fitzwater
NeXstar Pharmaceuticals

in reply to:
From: a03m at biologie.uni-bremen.de (Georg Kroeger)
Date: Wed, 03 Mar 1999 07:53:31 GMT

Hi all!
We have problems to precipitate small amounts of RNA (invisible
pellets got lost sometimes). Any hint for a nice coprecipitant would
be appreciated. By the way, at which point will I get rid of the
precipitant (70% EtOH whash?) again?
Thanks for your help!

Georg
=

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