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.