Automated sequencing

Michele M. Godlevski mam7746 at glaxo.com
Tue Aug 27 04:17:48 EST 1996


In article <4tni16$rq0 at net.bio.net>, Christiane Robbins
<crobbins at nchgr.nih.gov> wrote:

> HI there, I was wondering if someone has advice for the following problem:
> I often see a green haze on my 377 gels ,  I can still read my data but the
> gel is green. I have done everything from cleaning the plates with 3M HCL
> followed with ALconox, monitoring all sets of plates to see if it's only one
> set or one machine to no  avail. I still see this green haze and it's
not every
> gel. I would also like to know if anyone has a protocol for sequencing BAC's
> with custom primers using the FS-Terminator (ABI)?
> Thank you,
> Christiane Robbins
> 
> Christiane Robbins
> Bldg 49 Room 2C28
> 49 Convent Drive MSC 4431
> Bethesda, MD 20892-4431
> 
> Tel# 301-402-2534
> Fax#301-496-0474
> e-mail: crobbins at nchgr.nih.gov

Christiane,

To answer both of your questions...

We saw a yellow haze on our gels which turned out to be due in some cases
to dried acrylamide present on the amber colored pins on the machine. 
Essentially, the "crud" (as we termed it) was causing the plates to be out
of alignment and the result was a yellow haze.  On one machine, we had the
same problem repeatedly (minus any "crud") and found that the laser needed
to be re-aligned.

A client of ours is having some success with Bruce Birren's protocol for
BAC prepping. She restriction enzyme digests the DNA, and then uses
Birren's cycling conditions:
1. 96 degrees for 5 minutes
2. 96 degrees for 1 minute
3. 55 degrees for 1 minute
4. 60 degrees for 4 minutes
5. go to step 2 for 34 more times
6. 4 degrees hold


Michele Godlevski
Glaxo Wellcome
DNA Sequencing Facility
Research Triangle Park, North Carolina




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