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 
> unknown.
> 
> 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
purification blocks.


Michele Godlevski

Glaxo Wellcome Sequencing Core Facility
Research Traingle Park, North Carolina




More information about the Autoseq mailing list