Team:SDU-Denmark/labwork/Notebook/week7

From 2012.igem.org

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                                             <!----------7th WEEK---------->
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<p><b>20-08-2012 to 26-08-2012</b></p></br>
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<p>We made a gel of the SST 1 from earlier and cut out the slice.</br>
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<p><b>13-08-2012 to 19-08-2012</b><br></p>
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We did miniprep on the liquid cultures from the day before (SST 2 and FFT 9) and found out that there was no product of use from the FFT vial (bacteria must have grown badly) but excellent product from SST 2 (105,3 ηg/μL first time and 82,7 ηg/μL the second time) which was marked N1 and N2. </br>
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<h2>Preparing material for sequencing</h2>
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N1 was the first run of miniprep product and the N2 was the second. We made a gel where we ran the N1 SST on and cut out the smallest slice of the 2 fragments (approx. 1900 bp). The slice was extracted for DNA.</br></br>
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<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>
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We did miniprep on the liquid cultures from the day before (again) (on FFT 9) and found out that there was no product of use from the FFT vial (bacteria must have grown badly) only 4.0ηg/μL and 2.9ηg/μL in nanodrop. Therefore did we make new liquid cultures (FFT9).</br>
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From nanodrop:</br>
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We ran nanodrop on the SST PCR from yesterday and got about 412-420 ηg/μL from the nanodrop. </br>
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SST 1: 116ηg/μL</br>
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The liquid cultures form the 24th was taken out of the incubator, (after approximately 47 hours) and put in the refrigerator.</br></p>
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SST 2: 135ηg/μL</br>
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FFT 3: 57ηg/μL</br>
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FFT 3: 41ηg/μL</br></br>
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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>
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<p>The cultures from overnight had immensely low concentrations:</br>
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SST 1: 13ηg/μL</br>
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SST 2: 22ηg/μL</br>
 +
FFT 3: 23ηg/μL</br></br>
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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>
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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>
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The O.N. cultures was purified using the GeneJET Plasmid Miniprep Kit.</br></br> The results from nanodrop were excellent:</br>
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SST 1:        76 ηg/μL</br>
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SST 2: too low    ~25ηg/μL</br>
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FFT 3:            179 ηg/μL</br></br>
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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>
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For SST, the material in high concentration that were obtained monday were used,  
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and the material was sent for sequencing. A spare 5-7 μL were saved on freezer.</br></p>

Revision as of 11:14, 26 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

1st week 2nd week 3th week 4th week
5th week 6th week 7th week 8th week

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.

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