Barbara Simionati wrote:
> I was wondering if, among all those people that have purcased ABI 377
> sequencer 96-lane upgrade, there is someone that have succeded in using
> it without problems of mixing lanes, and if so what is your secret.
> I am also curious to know if someone have tested the MWG "automated comb
> loading for gel based DNA sequencing systems"
> (http://www.mwgbiotech.com/products/pcr/robots/index.htm), and your
> opinions on this new system.
> [...]
We haven't made extensive use of our 377 in 96 well mode yet. We've
primarily been using our 3700. But from what we've done, I'd say that using
the machine in 96 well mode should work fine--but there will be a learning
curve.
One big problem is tracking a gel that has a number of failed reactions
loaded. Especially if these reactions are next to each other. You can get
the sequence information, but it is difficult to be sure where the reaction
came from. I understand that PE-ABI is beta testing a kit that utilizes a
5th color labelled fragment to dope all your reactions before loading. Then
tracking should work unambiguously.
I saw the MWG robot at GSAC this year. Looks like it works --but keep in
mind you don't need a robot to utilize the porous paper (square-tooth)
combs. You can spot samples on them by hand with a pipet (that's what we
do.) I like the paper combs but don't think that they solve all gel loading
problems. They introduce their own set of problems. The main one is that
they are not very stiff. That means you have to make sure there are no
blockages (like small bits of acrylamide) in the well before you insert the
comb. Otherwise the teeth will bend and not make contact with the surface of
the gel. [I think they are just pourous nylon membrane of the right
thickness (like that used for blotting gels). I got my combs from The Gel
Company.]
Does everyone who uses the pourous paper combs use the water-load
procedure (that is, using water in the upper buffer chamber while
electrophoresing the samples into the gel. Then adding enough 10x buffer to
the upper buffer chamber to bring the buffer concentration up to a standard
level before continuing?)
Phillip San Miguel
Purdue Genomics Center
