sequence is blurry

Alexander Kraev kraev at bc.biol.ethz.ch
Wed Oct 9 05:32:05 EST 1996


In article <jmanalig-0810961652510001 at station87.medadmin.uiowa.edu>,
jmanalig at blue.weeg.uiowa.edu (jose manaligod md) wrote:

> I use FMC Long Range Gel mix for my 6% acrylamide gels and it has allowed
> me to have sharper bands and a longer read.  
> 
> 
> In article <genecutl-0410961813020001 at kosh-161b-3.berkeley.edu>,
> genecutl at mendel.berkeley.edu (gc) wrote:
> 
> > I've been having trouble reading my sequencing gels because the
> > bands tend to get blurry after about 60-100 bases from the bottom
> > and I can't tell if I'm looking at single bands or doublets.  I've 
> > been running 5% acrylamide gels at 35-40 watts.  The buffer is 
> > 1xTBE, except that I add sodium acetate to 1M in the bottom buffer
> > after the gel has run about half way.  Any suggestions on sharpening 
> > my bands would be grealy appreciated.
> > 

Blurry bands are more often than not a result of uneven heat dissipation.
This may happen because the power applied is excessive ( should be typically
<less> than 1 W per ml gel) or the gel is running under a strong air stream from
one side. Try placing the gel holder in a quiet place and clamp two alumina
plates to it, if your gel holder does not have any. In addition, with
"electrolyte gradient" gels, as the one mentioned above, the upper part is
heating up more than the lower as the salt gradient builds up, and
practically
all Joule heat is dissipated in the upper half, so the power should be
lower as with an equivalent gel without a gradient. The best way is to get
a thermocouple
and determine once the power setting that heats the upper part to 45-50 C
after 1 hour of run.
As for  the FMC gels, they do not give superior results relative to the EG
gels (though you do not need a gradient and provided that the power is
optimised), but they are far superior  to acrylamide for long runs and
long gels ( 60-80 cm).

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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