Laboratory Notebook

16-07-2012 to 22-07-2012

We still had no 1-SST colonies. Another PCR was made from the original cDNA, using various temperature steps. The pJET vector and 1-SST gene were run on the same gel and purified together on the same spincolumn during gel DNA extraction and mixed the elution buffer with ligation buffer and excess ligase in the final elution step.

The 1-SST colonies were incubated at 37C overnight on ampicillin agar medium plates.

A checkdigest was made on the 10 1-FFT liquid cultures with EcoRI and PstI. The resulting gel had two bands of the expected lengths for vector and insert. These liquid cultures were miniprepped and nanodropped:
Tube#3: 138,2ng/µl
Tube#9: 71,8ng/µl

Tube 3 contained a concentration suitable for sequencing. It was prepared and sent. We used the MWG sites primer design tool (http://www.eurofinsdna.com/) to design primers that anneal to the FFT gene. Seeing as the FFT gene is approximately 1800bp long the primers were designed to anneal between positions 450-500 and 950-1000 both in the forward direction, to be sure that the entire gene is sequenced.

The following day 1-SST cultures finaly showed some results, 6 colonies. They were transfered to liquid medium and incubated overnight./> The 6 colonies were miniprepped and nanodropped. They all showed concentrations of >60 ng/uL, some even around 160.

The following checkdigest showed no usable results.