Using Glycogen to prec DNA???

David Micklem drm21 at mole.bio.cam.ac.uk
Wed Nov 9 09:57:57 EST 1994


In article <39p5kb$o1c at nermal.cs.uoguelph.ca>, sannis at uoguelph.ca (Seanna
Annis) wrote:

> I am trying to purify a PCR fragments, around 600 bp from gel.  I can get 
> a decent amount out of the gel, but I lose it during the precipitation 
> step.  I want to use this product for direct cycle sequencing, so yeast 
> tRNA as an aid to precipitating the DNA was suggested to me to be a bad idea.
> I have heard of glycogen used to prec oligonucleotides.  Does anyone know 
> of the right concentration of glycogen to use.  And does it interfer with 
> sequencing afterwards?  Or does anyone know of another method to get a 
> better yield.  
> Thanks.  Seanna    (sannis at uoguelph.ca)

I use 2ul 10mg/ml glycogen (Boehringer) per 50-100ul aqueous regularly as
an _aid_ to precipitation - you still need the salt and alcohol!  I've
never had a problem with its interfering with any subsequent step, but I
haven't done cycle sequencing of PCR products. A major advantage IMO is
that because the glycogen coprecipitates, you can always SEE the pellet,
even if the amount of DNA is very small.  Beware though that the glycogen
makes the pellet slip/detach very easily, so you have to watch out that
you don't lose it when removing the SN/wash.

An alternative which I haven't (yet) tried is to use linear acrylamide. 
This has been discussed here before, including protocols for making/using
it.  Try searching the archives (eg by gopher to ftp.bio.indiana.edu) to
find those articles.

Good luck,

David


An alternative is to use linear

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)223 334129      Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)223 334089               
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