Q: purified primers for sequencing??????
szsudhi at mailbox.ucdavis.edu
Sat Apr 26 18:27:10 EST 1997
On Tue, 22 Apr 1997, Dr. Duncan Clark wrote:
> In article <5jhjih$el2 at light.nih.gov>, Bernard Murray
> <bernard at elsie.nci.nih.gov> writes
> >Maybe you can use very crude oligos for some applications
> >(I've never done it) but gel purified oligos have been fine for me for
> >PCR (Taq) and sequencing (Sequenase). Some methodologies may require
> >the full HPLC purification.
> We have an old 381A synthesiser and I use crude oligos for everything
> from PCR to sequencing to long PCR of 20kb. By crude I mean deprotected
> and just ethanol pptd. Running them on an FPLC every so often shows them
> to be pretty clean. Using purified compared to unpurified gave no
> difference for the PCR reactions we tested them on.
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
> Duncan Clark
> DNAmp Ltd.
> TEl/FAX 01252376288
I am not an expert on primer synthesis. The primers (unpurified or simply
undesalted) come with lot of salts which can be a pain in automated
sequencing or in site-directed mutagenesis. We get pur primers
synthesized in central facility at UC-Davis. The cheapest primer comes
with trityl group removed and lyophilized. For desalting we have to pay
extra. This we use directly for sequencing with sequenase kit (in our lab)
or using ABI sequencer at a central facility.
If the primer is not gel purified, it can be expected to have a very small
proportion of shorter chains. The coupling efficiency is pretty high these
days. The shorter chains correspond to a very small proportion and it is
unlikely to pose a problem in reading the gels.
I am quoting a reference for purifying deprotected oligodeoxynucleotides
wherein the authors have used n-butanol and the purification takes about
Sawadogo, M. and Van Dyke, M.W. 1991. A rapid method for the
purification of deprotected oligodeoxynucleotides. Nucleic Acids
Research, 19: 674.
I have used this method previously, when the oligo came as a solution in
ammonium hydroxide. Of course, that time I was doing sequencing manually
using sequenase 2.0 kit.
Dept. of Plant Pathology
Univ. of California,
Davis, CA 95616.
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