hplc of plasmids/small DNA fragments?

Richard_Heath heath at mbcf.stjude.org
Wed Nov 1 13:06:36 EST 1995


In article <stephan.witte-311095095102 at marvin.biologie.uni-konstanz.de>, stephan.witte at uni-konstanz.de (Stephan Witte) writes:
> Hi,
> 
> has anyone ever tried to purify plasmids/ezymatic digests of plasmids using
> hplc?
> What type of column and what type of solvents?
> 
> Thanks a lot for hints,
> 
> Stephan
> 
Yes, it can be done, and gives good yield of high quality DNA.  Pharmacia
published an article from my old lab on the subject - we used a Mono-Q 5/2
column (actually with an FPLC system, just watch the flow rate on HPLC!). 

The reference is :

Baldwin, J.E., Sutherland, J.D. and Martin, S.L. (1991) "Purification of single
stranded DNA and oligonucleotides by FPLC" _Science Tools_, v35, pp 4-5.

But we also used this for ds DNA - never actually tried it for digests:  you'd
probably need to cut a lot of DNA!  Basically, load the crude DNA in a 20mM
tris, pH 8.3, 1mM EDTA, 400mM NaCl, and elute with a gradient of NaCl up to 1M.
(Buffer for oligos was 20 mM NaOH, with NaCl from 0 to 2M)

If you need further info, phone Pharmacia and request the ref (I've lost my
copy), or e-mail me for more exact conditions (which means digging thru my
thesis!).

Best wishes,

Richard Heath
St Jude Children's Research Hospital  

> 
> 
> 
> *******************************************
> Stephan Witte
> Inst. of Immunology
> University of Constance
> Postfach 5560 M 660
> 78434 Konstanz
> Germany
> 
> Phone: +49-7531-88-2749
> Fax:     +49-7531-88-3102
> e-mail: stephan.witte at uni-konstanz.de
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