At 04:39 PM 3/28/2001 -0700, Linda Ballard wrote:
>on 3/28/01 9:50 AM, Sheryl Christofferson at sherylc@omrf.ouhsc.edu wrote:
>
>> Greetings-
>> I have a question from one of the researchers in
>> my group. He would like to run fluorescent samples
>> on a native sequencing gel: that is, one without
>> urea. Has anyone ever tried this on any automated
>> sequencer? I'm curious as to whether the analysis
>> parameters would have to be different.
>>
>> Thanks in advance-
>>
>> Sheryl Christofferson
>We have run native gels for SSCP and for large double stranded product.
>What exactly is he looking at?
>
>Linda
>
>Linda Wood Ballard
>Genomics Core Facility
>University of Utah
Since the topic of non-denaturing DNA gels has come up,
I was recently approached by a post-doc here who wanted to resolve a
223 bp from a 232 bp band (two different alleles from a multiplex PCR I
think). He asked me if he could do this on a 5% native polyacrylamide gel,
then stain the gel post-electrophoresis (he suggested with EtBr). Well,
the VERY short version of my long reply to him was that I would be
surprised if those two PCR products couldn't be resolved on a 5%
non-denaturing polyacrylamide gel, but the problem he would then face would
be in the staining and subsequent visualization of the DNA bands -
especially if the gel was large in size (as is typical for those vertical
gel apparatuses used in sequencing/fragment analysis), and not easily
accomodated on a typical uv light box. My parting advice was that it would
not be worth the trouble I envisioned he'd have to go through, and
recommended he consider using florescently-labeled primers in his PCR,
followed by resolution on urea gels. Could I have given this person more
"hope" that the non-denaturing method could be done with reasonable effort?
Bob Keefe
Robert G. Keefe, Ph.D.
Wadsworth Center/NYS Dept. of Health
Genomics Core Facility
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