User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/29

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pJET Sequence Results

Purpose: to evaluate sequences from pJET vector in order to determine success of LCR troubleshooting experiment

Results:

Sequences from the five pJET colonies were determined using sequencing primers just outside of the insertion site.

Colony 1 resulted in essentially no homologous sequences either with the forward or reverse primer. Colonies 2 and 3 showed homology with the ATF2 domain, which appears to have inserted on its own in both cases. Both colony 5 reactions failed. Colony 4 forward reaction gave week homology to part of the MV10 vector backbone??? and the reverse reaction was homologous to the ampicillin resistance marker and bla promoter? Possible cross reaction with sequencing primers on the pJET backbone or contamination.

Conclusions:

The LCR troubleshooting experiment yielded only negative results. Some of one part inserted into the pJET vector, but no annealed parts did. We had low transformation levels to begin with as well...probably time to move on to Gibson assembly.