840:153g:Projects/project19/2011/10/11

Today we prepared and ran a polymerase chain reaction (PCR) to amplify our gene fragments. We used the protocol that is on Dr. Schwekendiek's openwetware page because it is relatively simple and because it has been shown to work in past years and with other groups' DNA this year. The general setup and list of ingredients can be found here: <http://openwetware.org/wiki/840:153g:Materials#Standard_PCR_setup>.

We prepared premix for 9 PCR tubes. Of these, 6 were experimental. Each of the experimental tubes contained the master mix, both of our primers without biobrick eneds (forward and reverse, PCR-grade water, and our template DNA. We chose to use 4 microlitres of template DNA, at the high end of the range, in order to ensure a sufficient concentration of our gene.

The PCR had been set up to provide a gradient of annealing temperatures ranging from 38-61 degrees C across 8 columns. We placed our 6 experimental tubes such that they would anneal at a range of 42-61 degrees C. Because ideal annealing temperature varies depending on the DNA sample and especially the primers used, we expect to find a well that had maximum amplification. We tentatively expect this to occur at the lower end of the temperature gradient because the Tm of our primers are in the 51-53 degree C range.

After setup, the PCR was allowed to cycle for 3 hours, after which our instructor stored the amplified samples appropriately until our next session.