In my experience, PCR samples are much more hit and miss than cloned
samples. This is probably mostly due to the primer, which is usually a
gene-specific primer designed for its location and not for its efficiency
as a sequencing primer. Most customers use PCR primers for sequencing, so
if there are problems with the primer, the PCR product is less likely to be
a good template. However, when PCR samples do work, they often give much
better data than cloned DNA (I guess thats because they are less likely to
be contaminated with sugars, proteins, phenol etc.). My advice would be to
try sequencing with an internal primer, or re-do the PCR with new primers.
Yvonne
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Yvonne Foss
Molecular Biology Services
Department of Molecular Medicine
King's College School of Medicine and Dentistry
Rayne Institute, 123 Coldharbour Lane
London SE5 9NU
Tel 44 171 848 5901
Fax 44 171 733 3877