PCR amplification problems could use some help

Scott C Winkel scwinkel at prodigy.net
Sat Dec 4 10:04:23 EST 1999


I am working on an amplification of a gene that is 1109 bp. This particular
assay has worked fine in other labs but has yet to be successful within our
facility. Other assays of smaller base pair length are working great, but
for some unknown reason this larger size assay does not want to cooperate.
Our primers have been checked and seem to be working fine. Actually , all we
seem to be getting are primer dimers. We are using new dNTP's, taq +Ab,
Mg++, & 10x buffer. The protocol is identical to what has worked flawlessly
in other labs. The only variables that are different are the technician,
location, water, and machine. We are currently using the Perkins Elmer 9700
thermocycler.

If anyone has experience amplifying fragments this large I would love to
discuss the methods by which they are using.

Thanks

Scott

scwinkel at prodigy.net







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