Team:SDU-Denmark/labwork/Notebook/week6

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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week8">8th week      </a></span>    </regulartext></td>
     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week8">8th week      </a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week9">9th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week10">10th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week11">11th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week12">12th week</a></span>    </regulartext></td>
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                                             <!----------6th WEEK---------->
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                                             <!----------5th WEEK---------->
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<p> <b>06-08-2012 to 12-08-2012</b> </p>
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<p>
 +
The primers for mutagenesis arrived. We used the Stratagene quickchange lightning multi site-directed mutagenesis kit to perform the desired changes to our genes. Old templates were digested with DnpI. New colonies with the mutated plasmids were made through transformation of XL10-GOLD cells.<br/><br/>
 +
 
 +
The 1-SST plates showed colonies, but none for 1-FFT. Another mutagenesis and transformation, with slightly altered settings to fit the primers better, was performed on 1-FFT while liquid cultures were made from the SST colonies.<br/><br/>
 +
 
 +
The following day 1-FFT showed colonies, these were transferred to liquid cultures.<br/><br/>
 +
 
 +
The day after miniprepps were made for both 1-SST and 1-FFT, check digest was performed. While the gel was running, expected lengths were calculated based on the expected bands for both correct sequences and sequences still containing the undesired restriction sites.<br/><br/>
 +
 
 +
We had alot of succesful 1-SST colonies with none of the undesired restriction sites. Colony 1 and 2 were most awesome. Will be shipped for sequencing later on.<br/><br/>
 +
 
 +
The conclusion from the gel on 1-FFT was that the illegal XbaI restriction site was removed, the desired XbaI site was present, but unfortunately almost all the plasmids still contain the illegal EcoRI site except either of colony 3 and 4, which were mixed by accident the day before. Beforehand, we expected trouble with the Primer designed to remove the EcoRI site, because it had a lot of problematic alignments.<br/><br/>
 +
Following, another two batches of liquid colony was prepared (from the remains of colony 3 and 4) and incubated overnight, another two plates were prepared as a safety backup with the mixed liquid medium of the two colonies.<br/><br/>
 +
 
 +
The liquid cultures of FFT colonies 3 and 4 from the day before were purified, and digested with EcoRI+SpeI and XbaI+PstI, then run on a gel.<br/>
 +
In conclusion from the gel: though faint, colony 4 shows signs of the illegal EcoRI restriction site.<br/>
 +
Meanwhile, colony 3 was awesome. It will be prepared and sent for sequencing monday.<br/><br/>
 +
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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<p><b>13-08-2012 to 19-08-2012</b><br></p>
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From nanodrop the following day:<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>
+
SST 1: 116g/µL<br/>
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From nanodrop:</br>
+
SST 2: 135g/µL<br/>
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SST 1: 116ηg/μL</br>
+
FFT 3: 57g/µL<br/>
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SST 2: 135ηg/μL</br>
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FFT 3: 41g/µL<br/><br/>
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FFT 3: 57ηg/μL</br>
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FFT 3: 41ηg/μL</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>
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<p>The cultures from overnight had immensely low concentrations:</br>
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-
SST 1: 13ηg/μL</br>
+
-
SST 2: 22ηg/μL</br>
+
-
FFT 3: 23ηg/μL</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. The results from nanodrop were excellent:</br>
+
-
SST 1:        76 ηg/μL</br>
+
-
SST 2: too low    ~25ηg/μL</br>
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-
FFT 3:            179 ηg/μL</br>
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to reach a concentration 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, material from monday were used.</br>
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The material was sent for sequencing. A spare 5-7 μL were saved on freezer.</br></p>
+
 +
The concentrations from 1-FFT was too low for sequencing. New liquid cultures were prepared for overnight incubation.<br/>
 +
</p>

Latest revision as of 02:13, 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

06-08-2012 to 12-08-2012

The primers for mutagenesis arrived. We used the Stratagene quickchange lightning multi site-directed mutagenesis kit to perform the desired changes to our genes. Old templates were digested with DnpI. New colonies with the mutated plasmids were made through transformation of XL10-GOLD cells.

The 1-SST plates showed colonies, but none for 1-FFT. Another mutagenesis and transformation, with slightly altered settings to fit the primers better, was performed on 1-FFT while liquid cultures were made from the SST colonies.

The following day 1-FFT showed colonies, these were transferred to liquid cultures.

The day after miniprepps were made for both 1-SST and 1-FFT, check digest was performed. While the gel was running, expected lengths were calculated based on the expected bands for both correct sequences and sequences still containing the undesired restriction sites.

We had alot of succesful 1-SST colonies with none of the undesired restriction sites. Colony 1 and 2 were most awesome. Will be shipped for sequencing later on.

The conclusion from the gel on 1-FFT was that the illegal XbaI restriction site was removed, the desired XbaI site was present, but unfortunately almost all the plasmids still contain the illegal EcoRI site except either of colony 3 and 4, which were mixed by accident the day before. Beforehand, we expected trouble with the Primer designed to remove the EcoRI site, because it had a lot of problematic alignments.

Following, another two batches of liquid colony was prepared (from the remains of colony 3 and 4) and incubated overnight, another two plates were prepared as a safety backup with the mixed liquid medium of the two colonies.

The liquid cultures of FFT colonies 3 and 4 from the day before were purified, and digested with EcoRI+SpeI and XbaI+PstI, then run on a gel.
In conclusion from the gel: though faint, colony 4 shows signs of the illegal EcoRI restriction site.
Meanwhile, colony 3 was awesome. It will be prepared and sent for sequencing monday.

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 the following day:
SST 1: 116g/µL
SST 2: 135g/µL
FFT 3: 57g/µL
FFT 3: 41g/µL

The concentrations from 1-FFT was too low for sequencing. New liquid cultures were prepared for overnight incubation.

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