PCR SEQUENCING ADVICE

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
also.

: 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
luck.

Shiao Wang
University of Southern Mississippi




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