Sequencing near primer problems.

Jaideep Behari behari at bscr.uga.edu
Sun Nov 20 15:52:29 EST 1994


In article <1994Nov11.160401.1 at uwovax.uwo.ca>, 37_410 at uwovax.uwo.ca (OVan-Ham) writes:
>I'm trying to sequence an area close to my primer (<20bp) using the Promega
>fmol cycle sequencing kit.  The problem I've encountered is that I'm
>getting very strong bands running across all four sample lanes, both above
>and below the primer band.  These bands run about 24bp 5' to the primer
>(which, coincidentaly is my primer size) and can be very dark, or light.
>
>Has anyone else encountered this problem?  And more importantly, has
>anyone solved it?  If so please let me know what you've done.
>
>OVan-Ham at UWO.CA
>


I've been doing a lot of fmol sequencing myself but have never encountered
the bands you've described. However, we routinely use the terminal deoxy-
nucleotidyl transferase reaction to prevent bands in multiple lanes. I would
recommend that you take a look at : Fawcett,TW and Bartlett, SG, Biotechniques
Vol.9, No.1(1990) for a description of this method. Good luck.



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