Analysis of Library Clones 11-28-12
2014-08-29azim58 - Analysis of Library Clones 11-28-12
Analysis of 10 tumor and control clones revealed that only 1 clone had an
insert. The rest just had empty vector.
Sequencing results and analysis can be found here:
C:\kurt\storage\CIM Research Folder\DR\2012\11-28-12\human tumor library
sequence analysis
My hypothesis is that when we switched the using random hexamers the
strange Advantage 2 PCR protocol with an annealing temperature of 65 C
that was used with the original longer full length primers was no longer
appropriate for this PCR. I suspect that switching to a different
polymerase such as PrimeStar Max and decreasing the annealing temperature
to 55 C may fix many of these problems. These sequencing results may also
reveal why I have been obtaining such low transformation efficiencies. I
may have just been transforming a very small amount residual
non-linearized vector.