Shiao Y. Wang sywang at whale.st.usm.edu
Mon Sep 12 00:17:21 EST 1994

Edgar Salazar-Grueso (EFSG at biosci.bsd.uchicago.edu) wrote:
: I need advice. We just started isolating cDNAs from lambda phage that have
: been amplified by PCR for sequencing. We are using Gibco BRL's ds DNA PCR
: seqeuncing kit and a different primer than was used for the initial PCR.
: Although there are times we can get readable sequence, we still have two
: problems we have not been able to solve:  1) we get a lot of shadow bands (
: 2-4 lanes) & 2) stop artifact (all 4 lanes of equal intensity).   Some
: colleagues have suggested that the DNA might be dirty and that I should try
: cleaning it up further.  Others beleive that our PCR products might have 
: too much salt.  We have tried raising our annealing temperature up but this has
: given us +/- results.
: We isoalte our PCR products using millipore spin filters and then purify
: with PEG.

I've had good luck using one biotinylated primer and then using
streptavidin coated magnetic beads to purify ssDNA for sequencing. These
beads are available from Dynal (800-638-9416). They can provide protocols

: Please email me directly at efsg at biosci.bsd.uchcaigo.edu.

Let's stay on the net when possible. There may be others interested. Good

Shiao Wang
University of Southern Mississippi

More information about the Methods mailing list