Re: DNAsyn: purification of longmers

Richard T. Pon (rtpon@acs.ucalgary.ca)
Tue, 3 Feb 1998 09:11:40 -0700 (MST)

Hi
I think that the only method which can resolve such long sequences
is polyacrylamide gel electrophoresis. However, even with it, obtaining >
95% purity might be difficult, especially if you overload the gel. We
routinely use 16% polyacrylamide/7M urea gels in a modified Bio-Rad
Sequi-gen DNA sequencing cell (40 cm long, modified by using 1.5 mm thick
spacers). You can easily visualize the separation by UV shadowing and cut
out the full length product with a scapel. Extract the gel slices with
0.25M NaCl overnight and then desalt on a Sephadex (i.e. NAP-10) column.
We have used this method for oligo's as long as 170 bases.

On Mon, 2 Feb 1998, Mary Kay Dolejsi wrote:

>
> What is the "best" method for purifying full-length long oligomers- like 80
> - 85mers? A researcher here wants to make such a longmer, and have it >95%
> pure. Can solid-phase methods, like OPC really do this? Can IEX resolve at
> this length? Gel electrophoresis and elution?
>
> I appreciate any help you can give us. I know there was a recent
> discussion about a 45mer, but has anyone been working with longmers?
>

Richard T. Pon, Director E-mail: RTPon@acs.ucalgary.ca
University Core DNA Services Phone: (403) 220-4277
University Of Calgary Fax: (403) 283-4907
Please direct DNA synthesis or sequencing orders to DNALAB@acs.ucalgary.ca