fmol direct sequencing kit

Felipe N. Soto F-Soto at uiuc.edu
Wed Jun 28 11:44:24 EST 1995


I am trying to sequence directly PCR product for 18S using Promega's fmol
sequencing kit and I am having problems with bands in all four lanes. 
First I thought that my template was dirty so I ran a Nu Sieve gel, cut the
band out and purified the DNA using either the Elu-Quik kit of S&S,  or by
electroeulution.  The problem with the bands did not improve much.  My
primers are 16-21meres with GC content between 42 and 80%. I have used
temperature and time profiles which include 30secs at 95¡C followed by
30sec at 70¡C (30 cycles) and still I get bands in all four lanes.  My
primers are at most 2xdegenerate, and anyway, I do not see any difference
in the quality of the sequences produced by the exact primers and the
sequences produced with de degenerate primers.

Has anyone had this problem before, I will appreciate any suggestions.

Felipe N. Soto
Department of Entomology
505 S. Goodwin Ave.
Urbana, IL 61801
f-soto at uiuc.edu



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