T-blobs and bizzare fronts
Michele M. Godlevski
mam7746 at ussun2n.glaxo.com
Wed Jul 10 13:05:43 EST 1996
In article <4pic83$h11 at net.bio.net>, Ken Taylor <KTAYLO at scri.sari.ac.uk> wrote:
> Fellow sequencers,
> to continue the discussion started by Robot Boy
> (where did you get that name?) and Jenny Cassady, I've recently
> noticed the T-blobs for the first time. Quite a few gels have had
> this, though not all, and it began about three weeks ago.
> Is this happening to anyone else?
> On the Advanced sequencing course in Warrington we were told about
> this artefact. A T-blob in the second or third panel; ie between
> bases 200 and 320. On my (373 stretch) gels it's coming in about 340 -
> 360. I'm not sure if it is associated with a yellow haze
> (shouldn't it be red?), though I have seen this on gels, sometimes
> associated with microsatellites. I'll start checking all my images
> from now on.
> The word from ABI at the course was that the cause of the blob is
> Happy sequencing, Ken.
Ken and others plagued by T-blobs:
We have used routinely two methods of post-cycling purification here.
The best method (with no T-blobs and no 80-base front) is using the
A.G.T.C. blocks or Centri-Sep columns. The only case in which we have
seen T-blobs or 80-base fronts using these methods is when the column had
a hole in it or was too dry when the sample was added. This problem
appears more frequently with Centri-Seps than the A.G.T.C. blocks.
Unfortunately, though, the A.G.T.C. blocks are extraordinarily expensive.
Our second choice method is the A.B.I. MgCl2/70% EtOh precipitation with
the upside-down low-speed spin step. This added step effectively removes
T-blobs, but the 80 base front still remains.
I can send you a copy of that protocol if you like...or information on the
Glaxo Wellcome Sequencing Core Facility
Research Traingle Park, North Carolina
More information about the Autoseq