Team:Cambridge/Diary/Week 6

Monday

We are suffering from some setback on the PCR front. Tom and Emmy's PCR re-runs for the large vectors did not work despite lengthening the elongation time to 5 minutes per cycle. After sequence alignments we discovered that the primers may anneal to various sites on the primer, producing a range of undesired products. We will carry out touch down PCR tomorrow to make the annealing conditions more stringent.

PCR of the mOrange gene last Friday also yielded no visible products. Looking at the primers' designs again, we think it might be due to problems with the annealing temperature. Therefore we are setting PCR reactions across a temperature gradient today to find the optimum annealing temperature.

For the first time, we actually did some Gibson assembly in a realistic setting! We (hopefully) fused together the Mg2+ fragment purified last week, along with the riboswitch DNA purified two weeks ago. If all goes according to plan, the transformation on bacillus that we later performed should provide us with some responsive bacteria by tomorrow morning. If not, we'll try again in e.coli, and see if we can make a new biobrick from the riboswitch in this chassis.

Tuesday

A day of abject failure. Bacillus from yesterday has failed to grow at all. We're leaving them in the incubator for another day, but don't hold out much hope of getting any useful results.

The problem may have been our transformation, in which one of the steps may not have been at the correct 37 °C temperature. Consequently, we're re-running the experiment, as well as trying to get the plasmid working in some TOP10 e.coli.

Gel electrophoresis of the mOrange PCR products give no visible bands. Curiously, we also seem to have problems with our positive and negative controls.

Wednesday

Thursday

Friday