Team:SDU-Denmark/labwork/Notebook/week8

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<p><b>20-08-2012 to 26-08-2012</b></p></br>
<p><b>20-08-2012 to 26-08-2012</b></p></br>
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<h2> Miniprep and Nanodrop</h2>
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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>
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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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We recieved the Shine-dalgarno inducing primers and mutagenesis were performed on 1-SST and 1-FFT cultures, XL10-GOLD bacteria were transformed with these
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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 and cut out the smallest slice of the 2 fragments (approx. 1900 bp). The DNA was extracted form the gel.</br></br>
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and incubated overnight at 37C.
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We made a new miniprep on the liquid cultures from the day before (FFT 9) and found out that there were still no product of use from the FFT vial. With concentration of only 4.0ηg/μL and 2.9ηg/μL in nanodrop. Therefore we made a new liquid culture of FFT9.</br>
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We ran nanodrop on the SST PCR from yesterday and got about 412-420 ηg/μL concentration. </br>
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The following day we had colonies for both. These were transfered to liquid medium.
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The liquid SST cultures form earlier  was taken out of the incubator, (after approximately 47 hours) and put in the refrigerator.</br></p>
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The minipreps from the liquid cultures the following days yielded unexpectedly low results in the area around and below 10ng/uL. We were unsure why this
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occoured, but we began to expect the genes, which now contained bacterial RBS might be greatly inhibiting the bacterias growth. This does make sense, since
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inulin synthhesis would compete for sucrose with the bacterias sucrose metabolism, plus the synthesized inulin might be building up inside the bacteria. If
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this was the case, we needed to find a way around it in order to continue working efficiently with our genes.
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We did not have anything available that would be able to inhibit the pJET promoter, so we took a more simple approach: increase the incubation time with more medium.
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</p>

Revision as of 02:05, 27 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

1st week 2nd week 3th week 4th week
5th week 6th week 7th week 8th week
9th week 10th week 11th week 12th week

20-08-2012 to 26-08-2012


We recieved the Shine-dalgarno inducing primers and mutagenesis were performed on 1-SST and 1-FFT cultures, XL10-GOLD bacteria were transformed with these and incubated overnight at 37C. The following day we had colonies for both. These were transfered to liquid medium. The minipreps from the liquid cultures the following days yielded unexpectedly low results in the area around and below 10ng/uL. We were unsure why this occoured, but we began to expect the genes, which now contained bacterial RBS might be greatly inhibiting the bacterias growth. This does make sense, since inulin synthhesis would compete for sucrose with the bacterias sucrose metabolism, plus the synthesized inulin might be building up inside the bacteria. If this was the case, we needed to find a way around it in order to continue working efficiently with our genes. We did not have anything available that would be able to inhibit the pJET promoter, so we took a more simple approach: increase the incubation time with more medium.

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