Marking long range sequencing gels

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Fri Apr 7 03:55:57 EST 1995


In article <1995Apr6.183146.1 at molbiol.ox.ac.uk>, mcguire at molbiol.ox.ac.uk writes:
>
>Dear all,
>
>I am looking to specifically read a genetic marker located 800 bp
>from the nucleotide sequencing primers, the flanking sequence is
>not of particular importance. Besides manually producing a marker of
>this size, is there any company marketing DNA markers for sequencing
>gels, or any very slow running loading dye which could be used to cut
>out possible trial and error?
>
>Alternatively what's the general opinions of running 3.5%
>polyacrylamide gels?
>
You are not expected to read that far unless you specifically arrange for that.
The reliable systems all use gels that are AT LEAST 50 cm long. Even then you
would need to run your bands to the bottom to be well separated. Is it really
worth the effort? Can't you just make another primer closer? If not, consider
running a Hydrolink gel of 80 cm  ( can give details, if required ) length.
Otherwise look for automatic systems with long gels, such as LI_COR.
AS for 3.5% gels, they are difficult to handle on a conventional system.
They generally need to be covalently bound to the glass plate. Systems with
continuous detection or direct blotting electrophoresis handle these gels
easier, since they are kept within the plates all the time.
Good luck,

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
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CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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