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Team:SDU-Denmark/labwork/Notebook/week6
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Laboratory Notebook
13-08-2012 to 19-08-2012
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 50-100 ηg/μL we dilluted the 49μL buffer+plasmid with another 40μL buffer, leaving it at around 98ηg/μL.
For SST, material from monday were used.
The material was sent for sequencing. A spare 5-7 μL were saved on freezer.
