Team:SDU-Denmark/labwork/Notebook/week2

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<p> <b>09-07-2012 to 15-07-2012</b> </p>  
<p> <b>09-07-2012 to 15-07-2012</b> </p>  
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<h2>PCR of SST and FFT</h2>
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<p>PCR of SST and FFT:
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<p>
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When the primers arrived, 1-SST and 1-FFT were amplified with PCR(*polymerase chain reaction*) and isolated using gel electrophoresis.<br/>
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We did a <a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/PCR"><b>polymerase chain reaction</b></a> with two different settings; one where we used the recommended settings from the protocol and one where we specified the temperature for each cycle in the PCR which should be more optimal for the primers. <br>
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The gel showed clear bands near 1900bp, which is the expected lengths of our genes of interest.<br/>
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The genomic template we used was MCF7. The PCR samples were processed over night (O.N.).
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The PCR products from the gel were cut out and extracted using a PCR-extraction kit.<br/><br/>
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</p>
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<br/>
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The results from two succesful FFT gel bands on Nanodrop:<br/>
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345,5 ng/uL and 436,2 ng/uL<br/><br/>
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<h2>Gel for amplification of FFT and SST</h2>
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We did not get any bands for SST and made another PCR at different temperatures. We did get 2 weak bands for SST.<br/>
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<p>
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The results from Nanodrop:<br/>
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We did a gel on the PCR products from O.N. and got some usable bands from the FFT-gene. <br/>
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15,5ng/uL and 35,7ng/uL<br/><br/>
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We cut the band out of the gel a made a purification of it. We had some problems with the SST-gene and ended with no useable results. <br/>
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We made a NanoDrop of the purified FFT-gene and got a concentration of -1.0ng/μl. We measured a NanoDrop on the PCR products from the FFT-gene before we ran a gel on it to compare with the purified FFT-gene. This gave us a concentration at 103,2ng/μl. <br/>
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The results were not satisfying, but decent enough to move on to make extension PCR with the iGEM prefix and suffix overhangs in order to introduce the standardized restriction sites.<br/><br/>
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With this result we decided to repeat the purification of the FFT-gene.<br/>
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We tried to optimise the concentration of the FFT-gene from the gel and ended up with 13,8ng/μl determined by the NanoDrop. <br/>
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We decided to do a tranformation which was incubated O.N.
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<br/>
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We prepared another PCR for the SST-gene where we adjusted variables, like raising the temperature to 63°C for the annealing-step and adjusted the extension time. <br/>
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PCR was run overnight.
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<br/>
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We did not get immidiate good results, determined by Nanodrop analysis, so we experimented with different temperatures for the PCR annealing step. After some days of trial and error, we had decent nanodrop results, >10ng/uL, and we moved on to ligation and transformation.<br/><br/>
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<h2>Gel electrophoresis of PCR products from 10-07-12 and preparation of PCR on FFT and SST DNA</h2>
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We used the pJET1.2/BLUNT vector as transformation plasmid, since the labatory staff had good experience with this plasmid. It carries the ampicillin resistance and an EcoRV(blunt cut) restriction site within a toxin coding sequence with its own promoter and terminator.<br/><br/>
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<p>
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We ran a <a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/gel"><b>gel electrophoresis</b></a> the following day on the PCR products from the day before (the O.N. ones). After 1 hour at 100V we had fine separation of bands, and had very clear bands at the size where we had predicted our SST-gene to be: around 1900b. <br/>
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The bands were cut out and purified using a <a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/pcrgelclean"><b>PCR and gel clean-up</b></a>. <br/>
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The samples from the gel extraction were tested with Nanodrop which gave us uncertain results.
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We decided to make a transformation with our SST-genes even though the Nanodrop was a bit ambiguous.<br/>
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<br/>
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We made PCR reaction on 16 different colonies from the FFT agar plate from the day before. For the first 8 tubes, we used primers standardized for the pJET1.2/BLUNT and the last 8 tubes we used the primers for extended FFT.<br/>
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We ran a gel on the <b>PCR products:</b> <br/>
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PCR 1-8,  primer 3 and 4 and  PCR 9-16,  primer 354 and 355 (pJET1.2_for &  pJET1.2_rev).<br/>
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The gel gave inconclusive results for both sets of primers. <br/>
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<br/>
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From the agar plate with FFT-ex clones, we took out 10 random colonies and put into liquid LB and put in the incubator at 37°C O.N. <br/>
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The SST plate was also incubated O.N.
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The blunt ligation was immidiately followed by transformation and the cultures were incubated overnight on ampicilin agar media plates.<br/><br/>
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We had succes with 10 1-FFT colonies, but no luck with 1-SST, so we had to do another ligation and transformation. FFT colonies were transferred to liquid ampicillin containing medium and incubated overnight with SST ampicillin agar plates.<br/>
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</p>
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Latest revision as of 02:04, 27 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

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

09-07-2012 to 15-07-2012

PCR of SST and FFT: When the primers arrived, 1-SST and 1-FFT were amplified with PCR(*polymerase chain reaction*) and isolated using gel electrophoresis.
The gel showed clear bands near 1900bp, which is the expected lengths of our genes of interest.
The PCR products from the gel were cut out and extracted using a PCR-extraction kit.

The results from two succesful FFT gel bands on Nanodrop:
345,5 ng/uL and 436,2 ng/uL

We did not get any bands for SST and made another PCR at different temperatures. We did get 2 weak bands for SST.
The results from Nanodrop:
15,5ng/uL and 35,7ng/uL

The results were not satisfying, but decent enough to move on to make extension PCR with the iGEM prefix and suffix overhangs in order to introduce the standardized restriction sites.

We did not get immidiate good results, determined by Nanodrop analysis, so we experimented with different temperatures for the PCR annealing step. After some days of trial and error, we had decent nanodrop results, >10ng/uL, and we moved on to ligation and transformation.

We used the pJET1.2/BLUNT vector as transformation plasmid, since the labatory staff had good experience with this plasmid. It carries the ampicillin resistance and an EcoRV(blunt cut) restriction site within a toxin coding sequence with its own promoter and terminator.

The blunt ligation was immidiately followed by transformation and the cultures were incubated overnight on ampicilin agar media plates.

We had succes with 10 1-FFT colonies, but no luck with 1-SST, so we had to do another ligation and transformation. FFT colonies were transferred to liquid ampicillin containing medium and incubated overnight with SST ampicillin agar plates.

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