The iGEM Team of University of Southern Denmark 2012 igem.sdu.2012@gmail.com

Laboratory Notebook

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13-08-2012 to 19-08-2012

Preparing material for sequencing

The concentration of the FFT plasmid was low. Too low to be sent for sequencing, so a liquid culture was prepared for overnight incubation in the hopes of obtaining a higher concentration.

From nanodrop:
SST 1: 116ηg/μL
SST 2: 135ηg/μL
FFT 3: 57ηg/μL
FFT 3: 41ηg/μL

We need to address a problem with our genes. The transition from eukaryotic cells to bacterial cells has come with an untill now unforseen complication: the ribosomal binding site for eukaryotic cells(Kozak sequence), does not bind bacterial ribosomes, so we need to insert the bacterial version(Shine-Dalgarno).

LARS SKAL UDDYBE!!

The cultures from overnight had immensely low concentrations:
SST 1: 13ηg/μL
SST 2: 22ηg/μL
FFT 3: 23ηg/μL

A new liquid culture was prepared from the former liquid cultures for overnight incubation. To get high concentrations, they were incubated at 37°C for 22 hours.
New primers were designed and ordered to accommodate the changes needed for the Shine-Dalgarno sequence in appropriate distance from the start codon (6-7 nucleotides)
The O.N. cultures was purified using the GeneJET Plasmid Miniprep Kit.

The results from nanodrop were excellent:
SST 1: 76 ηg/μL
SST 2: too low ~25ηg/μL
FFT 3: 179 ηg/μL

To reach a concentration of 50-100 ηg/μL we dilluted the 49μL buffer+plasmid with another 40μL buffer, leaving it at around 98ηg/μL.
For SST, the material in high concentration that were obtained monday were used, and the material was sent for sequencing. A spare 5-7 μL were saved on freezer.