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Team:SDU-Denmark/labwork/Notebook/week6
From 2012.igem.org
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<p><b>13-08-2012 to 19-08-2012</b><br></p> | <p><b>13-08-2012 to 19-08-2012</b><br></p> | ||
| - | <p>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.</br> | + | <p>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.</br></br> |
From nanodrop:</br> | From nanodrop:</br> | ||
SST 1: 116ηg/μL</br> | SST 1: 116ηg/μL</br> | ||
SST 2: 135ηg/μL</br> | SST 2: 135ηg/μL</br> | ||
FFT 3: 57ηg/μL</br> | FFT 3: 57ηg/μL</br> | ||
| - | FFT 3: 41ηg/μL</br> | + | FFT 3: 41ηg/μL</br></br> |
| - | 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). <h1>LARS SKAL UDDYBE!!</h1></br> | + | 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). <h1>LARS SKAL UDDYBE!!</h1></br></br> |
<p>The cultures from overnight had immensely low concentrations:</br> | <p>The cultures from overnight had immensely low concentrations:</br> | ||
SST 1: 13ηg/μL</br> | SST 1: 13ηg/μL</br> | ||
SST 2: 22ηg/μL</br> | SST 2: 22ηg/μL</br> | ||
| - | FFT 3: 23ηg/μL</br> | + | FFT 3: 23ηg/μL</br></br> |
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.</br> | 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.</br> | ||
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)</br> | 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)</br> | ||
| - | The O.N. cultures was purified using the GeneJET Plasmid Miniprep Kit. The results from nanodrop were excellent:</br> | + | The O.N. cultures was purified using the GeneJET Plasmid Miniprep Kit.</br></br> The results from nanodrop were excellent:</br> |
SST 1: 76 ηg/μL</br> | SST 1: 76 ηg/μL</br> | ||
SST 2: too low ~25ηg/μL</br> | SST 2: too low ~25ηg/μL</br> | ||
| - | FFT 3: 179 ηg/μL</br> | + | FFT 3: 179 ηg/μL</br></br> |
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.</br> | 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.</br> | ||
For SST, the material in high concentration that were obtained monday were used, | For SST, the material in high concentration that were obtained monday were used, | ||
Revision as of 11:06, 26 September 2012
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 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.
