Team:SDU-Denmark/labwork/Notebook

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Lab Work
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     <div id="contenta">
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<h1>Laboratory Notebook</h1>
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--->
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Here you will find the log book for the procedures carried out in the laboratory, starting from week 27.   
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<h2>Laboratory Notebook</h2>
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<p>
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Here you find the log book for the procedures carried out in the laboratory, starting from week 27.   
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</p>
</p>
<table border="1" bordercolor="#FE1919" style="background-color:#EDEDED" width="100%" cellpadding="3" cellspacing="3">
<table border="1" bordercolor="#FE1919" style="background-color:#EDEDED" width="100%" cellpadding="3" cellspacing="3">
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<td><regulartext>
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<td id="tablestyle1"><regulartext>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook"><b>1st week</b>     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook"><b>1st week</b></a></span>    </regulartext></td>
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<td><regulartext>
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     <span><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week2">2nd week     </a></span>     </regulartext></td>
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     <span><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week2">2nd week</a></span></regulartext></td>
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<td><regulartext>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week3">3th week     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week3">3rd week</a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week4">4th week     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week4">4th week</a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week5">5th week     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week5">5th week</a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week6">6th week     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week6">6th week</a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week7">7th week     </a></span>    </regulartext></td>
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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week7">7th week</a></span>    </regulartext></td>
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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>
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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>
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<td id="tablestyle1"><regulartext>
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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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<td id="tablestyle1"><regulartext>
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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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<td id="tablestyle1"><regulartext>
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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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<td id="tablestyle1"><regulartext>
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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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</tr>
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</table>  
                                             <!----------1ST WEEK---------->
                                             <!----------1ST WEEK---------->
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<p> <b>05-07-2012:</b> </p>
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<p> <b>02-07-2012  to  08-07-2012</b> </p>
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<b>Securing plant material</b> <br/>  
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<p>The first week in lab started shortly after our exams. We had a goal, but were still uncertain about how to get there. We needed to get our hands on the coding sequence for a naturaly occouring inulin producing unit and there were several possible ways to get it.</br>  
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Plant material from Jerusalem artichoke (<i>Helianthus tuberosus</i>) were secured from a nearby plant nursery (Langeskov Planteskole).
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We could synthesise it, isolate it from a plant with a suitible kit or something less handy. Since the iGEM project is a learning experience to us, we chose something less handy
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<p>
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and designed our own experiment to isolate the mRNA from a suitable plant, using only the equipment and materials our lab had acces to from the beginning. <br/>
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<p> <b>06-07-2012:</b> </p>
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After having sought out information on the net, we decided on the plant Helianthus Tuberosus, also known as Jerusalem artichoke, to be the source of the coding sequence(s). Helianthus Tuberosus has a two-part inulin synthesis consisting of 1-SST and 1-FFT. Both genes are needed for the complete synthesis and thus both needed to be aquired. <br/>
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<b>Isolation of mRNA from Helianthus tuberosus </b> <br/>  
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During the summer, June-July-August, the Helianthus Tuberosus is growing and producing alot of inulin in the tubers of the plant. We took advantage of that and used our newly developed plant mRNA isolation protocol to isolate mRNA from the tubers.<br/>
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(The procedure we used for mRNA isolation, is a modified version of the Qiagen RNeasy protocol for plant mRNA isolation.)<br/>
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The process involved cutting up the tubers, flash-freezing with liquid nitrogen to halt RNase activity, which is VERY important when isolating RNA. Following, the plant material was grinded into fine dust while kept frozen to keep the plant cell walls rigid in an attempt to destroy them and release the mRNA and dissolved in RTL buffer. <br/>
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Small tubers from <i>Helianthus tuberosus</i> were washed with untreated water and cut in rough slices with an everyday boxcutter.<br/>
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The solution was treated with ultrasound to homogenize it to further disrupt the remaining cell wall. Cell wall and larger organelles were pelleted by centrifuge. From here on a mRNA isolation kit for mamalian cells was used to extract the RNA from the remaining solution.<br/>
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The slices were distributed in cryotubes and weighed, the weights were noted down, and submerged in liquid nitrogen(flash-freeze at -196 °C). Flash-freezing is necessary to halt all RNase activity. <br/>
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See *mRNA isolation protocol* for further details.<br/>
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RLT buffer is mixed at this point, for later use. For each mL RLT, add uL beta-mercaptoethanol. Do this in a fume cabinet, as mercaptoethanol smells really bad and is toxic!. <br/>
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Using reverse transcriptase we were able to convert the isolated mRNA to cDNA.<br/><br/>
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After 20 minutes 600mg is extracted from the cryotubes, the rest is stored for later use. The 600mg plant material is grinded to a fine dust using a mortar. <br/>
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It is important to keep the plant material frozen during the grinding, to keep the cell wall rigid enough to destroy it.
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The now isolated mRNA solutions were measured on a Nanodrop to ascertain their concentrations:<br/>
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The plant powder is transferred to a tube and dissolved in RLT buffer(450uL buffer op 100mg plant material). <br/>  
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-
We used approx. 3mL for 600mg material.The solution is mixed using a vortex mixer.<br/>
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Control: 627 ng/uL<br/>
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500 uL of the solution is transfered to a 2mL treated with ultrasound to homogenize the content and further disrupt the cell wall. 500 uL is transfered to a 2mL tube as a control. <br/>
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Sonic: 747 ng/uL <br/><br/>
-
The samples are centrifuged at 14.500 rpm for 60 seconds to pellet out large organelles. <br/>
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-
0,5 vol. 96-100% ethanol is added(250uL) to the sample, mixed by pipette. Transfer the samples to spin-column (Qiagen RNeasy mini kit, for RNA isolation from animal cells) in a 2mL sampletube, next:
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Furthermmore we designed primers that would anneal to the ends of the 1-SST and 1-FFT coding sequences, with the help of Steffen Smidth, and ordered them home.<br/>
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<p>
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-spin at 10.000rmp for 15s, discard flowthrough
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<center><img src="https://static.igem.org/mediawiki/igem.org/a/ae/IGEM098.jpg" width="100%" /></center>
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add 700uL RW1 til spin column <br/>
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-spin at 10.000rmp for 15s, discard flowthrough
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add 500uL RPE buffer til spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<br/>
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-spin at 10.000rmp for 15s, discard flowthrough
+
</p>
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add another 500uL RPE buffer to the spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<br/>
+
 
-
-spin at 10.000rmp for 15s, discard flowthrough
+
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Transfer the columns to new 2mL centrifuge tubes. Centrifuge at full speed for 1 min.
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-
Transfer spin columns to new 1.5mL sample tubes <br/>
+
 
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-add 30-50uL RNase-free water <br/>
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-spin at 10.000rpm for 60 seconds <br/>
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Repeat the prior step once. <br/>
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The mRNA is now isolated in the 60-100uL at the bottom of the collection tubes
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<p>
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The solutions were tested on <b>Nanodrop</b>: <br/>
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Control: 627 ng/uL <br/>
+
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Sonic: 747 ng/uL <br/>
+
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<p>
+
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As expected, disruption of the cell wall by ultrasound resulted in a higher yield.
+
 
 
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Latest revision as of 02:03, 27 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

Here you will find the log book for the procedures carried out in the laboratory, starting from week 27.

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

02-07-2012 to 08-07-2012

The first week in lab started shortly after our exams. We had a goal, but were still uncertain about how to get there. We needed to get our hands on the coding sequence for a naturaly occouring inulin producing unit and there were several possible ways to get it.
We could synthesise it, isolate it from a plant with a suitible kit or something less handy. Since the iGEM project is a learning experience to us, we chose something less handy and designed our own experiment to isolate the mRNA from a suitable plant, using only the equipment and materials our lab had acces to from the beginning.
After having sought out information on the net, we decided on the plant Helianthus Tuberosus, also known as Jerusalem artichoke, to be the source of the coding sequence(s). Helianthus Tuberosus has a two-part inulin synthesis consisting of 1-SST and 1-FFT. Both genes are needed for the complete synthesis and thus both needed to be aquired.
During the summer, June-July-August, the Helianthus Tuberosus is growing and producing alot of inulin in the tubers of the plant. We took advantage of that and used our newly developed plant mRNA isolation protocol to isolate mRNA from the tubers.
The process involved cutting up the tubers, flash-freezing with liquid nitrogen to halt RNase activity, which is VERY important when isolating RNA. Following, the plant material was grinded into fine dust while kept frozen to keep the plant cell walls rigid in an attempt to destroy them and release the mRNA and dissolved in RTL buffer.
The solution was treated with ultrasound to homogenize it to further disrupt the remaining cell wall. Cell wall and larger organelles were pelleted by centrifuge. From here on a mRNA isolation kit for mamalian cells was used to extract the RNA from the remaining solution.
See *mRNA isolation protocol* for further details.
Using reverse transcriptase we were able to convert the isolated mRNA to cDNA.

The now isolated mRNA solutions were measured on a Nanodrop to ascertain their concentrations:
Control: 627 ng/uL
Sonic: 747 ng/uL

Furthermmore we designed primers that would anneal to the ends of the 1-SST and 1-FFT coding sequences, with the help of Steffen Smidth, and ordered them home.

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