problems sequencing plasmids prepped from TG1

Wolfgang Schechinger novalidaddress at nurfuerspam.de
Tue Apr 25 08:41:54 EST 2006


Dear Experts,

we try to get sequences clones from a phage display experiment grown in
TG1.
from about 60 clones we had sent to a sequencing firm, only 9 yielded
data. the company said the DNA was in best condition. the plasmids were
prepped with a quiagen miniprep kit.

we had tested several samples by PCR (one of the primers is th e
sequencing primer,no problems, if you can compare PCR and sequencing at
all), and we find a band with the size of the expected insert in
restriction digests.

we are quite clueless now.

our next options would be re-transforming the plasmid in DH5alpa and
sending samples to some more labs.

any ideas what could be wrong and what to do?

Wolfgang Schechinger
Endocrine Research Lab
Bochum University Hospital "Bergmannsheil"



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