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cDNA precipitation without coprecipitation of primers

cDNA precipitation without coprecipitation of primers - Protocols and Methods Forum

cDNA precipitation without coprecipitation of primers - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 03-05-2007, 10:21 AM
Simone Marker
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Default cDNA precipitation without coprecipitation of primers



Hi,

I need to remove a 47bp primer from an RT reaction, i.e. I want to
precipitate the synthesised cDNA without the coprecipitation of the primer.
The easiest way would be the purification by spin coulmn, but the amount of
my cDNA is very low, so that I'm afraid to lose the cDNA on the column. Does
anyone know about precipitation conditions (isoprop/ethanol,different salts)
that eclude the coprecipitation of smaller nucleic acid fragments? (my cDNA
is at least one kb)

and in general: does anyone know if cDNA requires different precipitation
conditions than genomic DNA or RNA?


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Old 03-05-2007, 08:36 PM
Bas Jansen
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Default cDNA precipitation without coprecipitation of primers

In article <esgqv2$bu8$[Only registered users see links. ]-kl.de>,
"Simone Marker" <[Only registered users see links. ]-kl.de> wrote:


I remember trying to get rid of SAGE primers of around 20 nt by
precipitating DNA and a little ammonium acetate (7.5 M stock, add 1/10
v/v) and adding 100% ethanol to an end concentration of 40%. Worked like
a charm.
Just try to precipitate a few hundred nanograms of your 47 nt oligo in a
concentration range of ethanol, from 20% up to 70%, and run it on a
polyacrylamide gel to see whether it is still there.

Bas
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