Laboratory Notebook

10-09-2012 to 16-09-2012

We only had problems since we started working with the iGEM plasmids. For some reason the ligation will not work. Meanwhile, the iGEM standard primers VF2 and VR we recieved from the former iGEM team seem ineffective and produce strange results. We expect this is because they are more than 3 years old. New were ordered home.

We are running short on time and with the final days drawing close, we decided to start preparing characterization of the genes in the pJET vector, while attempting to ligate the sequences into pSB1C3.

With nothing that faintly looked like results at the end of the week, we took a different approach and ran a large scale PCR at a wide range of temperatures in order to hit the optimal conditions for our and the iGEM primers. A PCR program containing 4 different annealing temperatures proved succesful. It was designed to "lure" the primers to bind, by starting out at denaturation temperature, then dropping below 50C to promote fast binding of our long 70bp primers and slowly raising the temperature to sequence melting temperature to avoid unspecific binding before the elongation step.

With this new PCR program we were able to reproduce high concentrations of both 1-SST and 1-FFT for ligation into pSB1C3.