Team:SDU-Denmark/labwork/Notebook

From 2012.igem.org

(Difference between revisions)
 
(14 intermediate revisions not shown)
Line 218: Line 218:
<!--\\\\ ACCORDION MENU ////-->
<!--\\\\ ACCORDION MENU ////-->
-
<iframe src="https://2012.igem.org/Team:SDU-Denmark/menu" frameborder="0" height="500" width="200" scrolling="no"  ALLOWTRANSPARENCY="true">
+
<iframe src="https://2012.igem.org/Team:SDU-Denmark/menu" frameborder="0" height="1000" width="200" scrolling="no"  ALLOWTRANSPARENCY="true">
   <p>Your browser does not support iframes.</p>
   <p>Your browser does not support iframes.</p>
</iframe>
</iframe>
Line 241: Line 241:
<!-- /// velkomst ////-->
<!-- /// velkomst ////-->
-
<h2>Laboratory Notebook</h2>
+
<h1>Laboratory Notebook</h1>
-
Here you find the log book for the procedures carried out in the laboratory, starting from week 27.   
+
Here you will find the log book for the procedures carried out in the laboratory, starting from week 27.   
</p>
</p>
-
<table >
+
<table border="1" bordercolor="#FE1919" style="background-color:#EDEDED" width="100%" cellpadding="3" cellspacing="3">
<tr>
<tr>
<td id="tablestyle1"><regulartext>
<td id="tablestyle1"><regulartext>
Line 272: Line 272:
<td id="tablestyle1"><regulartext>
<td id="tablestyle1"><regulartext>
     <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>
 +
</tr>
 +
<tr>
 +
<td id="tablestyle1"><regulartext>
 +
    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week9">9th week</a></span>    </regulartext></td>
 +
 +
<td id="tablestyle1"><regulartext>
 +
    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week10">10th week</a></span>    </regulartext></td>
 +
 +
<td id="tablestyle1"><regulartext>
 +
    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week11">11th week</a></span>    </regulartext></td>
 +
 +
<td id="tablestyle1"><regulartext>
 +
    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week12">12th week</a></span>    </regulartext></td>
</tr>
</tr>
</table>  
</table>  
Line 279: Line 292:
<p> <b>02-07-2012  to  08-07-2012</b> </p>
<p> <b>02-07-2012  to  08-07-2012</b> </p>
-
<p>Plant material from the Jerusalem artichoke (<a href="http://en.wikipedia.org/wiki/Jerusalem_artichoke"><i>Helianthus tuberosus</i></a>) were secured from a nearby plant nursery(<a href="http://www.langeskov-planteskole.dk/"><i>Langeskov Planteskole</i></a>). The </p>
+
<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>
 +
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. <br/>
 +
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/>
 +
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/>
 +
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/>
 +
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/>
 +
See *mRNA isolation protocol* for further details.<br/>
 +
Using reverse transcriptase we were able to convert the isolated mRNA to cDNA.<br/><br/>
-
<p>
+
The now isolated mRNA solutions were measured on a Nanodrop to ascertain their concentrations:<br/>
-
The procedure used to isolate mRNA from Helianthus tuberosus, was a modified version of the Qiagen RNeasy protocol for plant mRNA isolation. See <a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols"><b>mRNA isolation</b></a> protocol for details<br/>
+
 
-
Plant material form <i>Helianthus tuberosus</i> was cut into pieces and stored in liquid nitrogen(flash-freeze at -196 °C)to halt all RNase activity. RLT buffer is mixed at this point, for later use. <br/>   
+
Control: 627 ng/uL<br/>
-
After 20 minutes of freezing time the plant material was grinded into fine dust and dissolved in RTL buffer. The solution was treated with ultrasound to homogenize it, and to further disrupt the cell wall. <br/>
+
Sonic: 747 ng/uL <br/><br/>
-
After centrifugation, Ethanol was added to the solution and the sample was transfered to a spin-column from the Qiagen RNeasy mini kit. The kit i usually used for RNA isolation from animal cells was used (see protocol for details). 
+
   
-
The now isolated mRNA solutions were measured on a <b>Nanodrop</b> to ascertain their concentrations:  
+
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/>
-
<p>
+
 
-
Control: 627 ng/uL <br/>
+
<center><img src="https://static.igem.org/mediawiki/igem.org/a/ae/IGEM098.jpg" width="100%" /></center>
-
Sonic: 747 ng/uL <br/>
+
 
-
<p>
+
 
 +
 
 +
</p>
    
    
   
   

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.