Date: Thu, 09 Jan 1997 15:39:13 -0700 (MST)
From: "Terry Mulcahy 272-5792; fax 272-9107" <TMULCAHY@medusa.unm.edu>
Subject: DNA Synthesis
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Date: Mon, 11 Nov 1996 17:02:35 -0800 (PST)
From: Deb McMillen <mcmillen@morel.uoregon.edu>
Subject: Re: Purification of ds 15-mer oligonucleotide
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Old-To: Amos Heckendorf <nestgrp@world.std.com>
Amos,
Hello,
We are trying to obtain a purified synthetic DNA oligomer. We have two
15 mers one of which is labeled with rhodamine. We want to obtain the
paired product. Lara (the grad student with this project) has run the
product on a gel and but gets low yield when she ethanol precipitates the
double stranded product. I've given her another protocol to follow using
a Seppak cartridge to get the DNA from the gel material. I've come
across several ideas in Maniatis, such as electrophoresing the band into
a DEAE-cellulose membrane and then eluting off that membrane. Or using
Pharmacia's DEAE-Sephacel to clean up the DNA out of the gel slice. Or the
crude reaction mixture could be cleaned up on a hydroxyapatite column:
sounds like it would bind the
double-stranded and single stranded DNA products--and different levels of
phosphate buffer would elute first the single stranded product and then
the double stranded product. I was also think that we could try the
traditional RP-HPLC mobile phase system, 0.1 M TEAC pH6.0 with an
acetonitrile gradient on our Vydac C4 column. Do you have any other
ideas? Which do you think is better? Here are the oligomer sequences:
gggctataaaagggg - rhodamine
ccccttttatagccc
Thanks for your help,
Deb McMillen
Institute of Molecular Biology
University of Oregon