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Team:SDU-Denmark/labwork/Notebook/week7
From 2012.igem.org
Laboratory Notebook
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.
