Team:British Columbia/Notebook/Week 4

From 2011.igem.org

In a group, Vicki, Marianne, Gurpal and Jacob attempted to do a site-directed mutagenesis on June 28th. Gurpal chose to do his on the PgxeTPS-Cin synthase gene. The goal was to remove an internal cut-site. He prepared 2 samples and a water control. ThermoPol was used as a buffer and PFU were used. After adding the forward and reverse primers (and other constituents), we each ended up with ~ 25 microL samples in PCR tubes. We set our samples to run in a thermocycler for about 4 hours with specified conditions. When the cycling was completed, DPN1 was added to each PCR tube and then the tubes were incubated overnight at 37 degrees Celsius.

In order to test our site-directed mutagenesis experiment, we prepared gels and preformed a gel electrophoresis. We did this one hour after the PCR tubes were incubating at 37 degrees Celsius. Unfortunately, our gel showed that our site directed mutagenesis did not work. But, each of us learned what not to do for next time which is always good.