Long Ranger seq. gel solution.

David Johnstondaj daj at nhm.ic.ac.uk
Thu Jul 15 06:13:50 EST 1993


On 14 Jul 93 14:11:38 GMT,
  <cxxder02 at uctvax.uct.ac.za> writes:

>Hi there,              
>I am at present using  the Hydrolink "Long Ranger" gel solution for 
>sequencing gels and am  having problems with getting fuzzy  bands towards 
>the top of medium to long reads. I've checked all the recommendations in the
>trouble shooting guide but to no avail.  Is anybody else using this gel 
>concentrate having  or had similar problems and if so would I be better off
>going back to using the conventional acrylamide gels.

We have been getting nice, sharp bands and readings up to approx 650 bases 
from the primer (best to date and miles better than anything we have 
acheived using standard acrylamide) using LONG RANGER on 0.2mm, 60cm gels. 
We run them real cool/slow (40ish degrees C) with 1.2XTBE in the gel, 0.
6XTBE in the tanks. We use a 0.18mm OD sequencing pipette to very gently 
load the samples as a sharp band right at the bottom of the sharkstooth 
well rather than let it sink down (a steady hand job if ever ther was one). 
We also fix/wash our gels in 3L of 10% methanol 10% glacial acetic (despite 
the standard protocol saying you don't need too - we have never had a gel 
we could afford to loose by trying it out). Incidentally the gels really do 
seem to be tougher than standard acrylamide ones which makes handling 0.
2mm gels less daunting (all I need to do is to find an easy way of 
pouring them - I would have thought that the thinner the gel, the easier it 
would be to pour as capillary action would give you more of a helping 
hand - this does not appear to be the case and they fill incredibly 
slowly - chill your gel mix before pouring)

This being said, it doesn't always give brilliant results, sharpness 
can vary from gel to gel and from reaction to reaction within a gel so my 
suspicion is that something in the reactions is causing the fuzzyness. 
Anyone got any ideas what?

Cheers
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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