Team:DTU-Denmarkgogo

From 2014.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 33: Line 33:
<td>
<td>
-
<center><a href="h"><img src="https://static.igem.org/mediawiki/2014/8/8e/PCR_PIC.png" width="942" height="200"/> </a> </center>
+
<center><a href="h"><img src="https://static.igem.org/mediawiki/2014/8/8e/PCR_PIC.png" width="942" height="180"/> </a> </center>
<tr>
<tr>
Line 40: Line 40:
-
<ol>
+
<ul>
-
<li>Add one ml sterile 50% glycerol to a cryotube.</li>
+
<li>Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.</li>
-
<li>Add one ml overnight culture to a cryotube from step 1</li>
+
<li>The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.</li>
-
<li>Store at -80 °C</li>
+
<li>Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.</li>
-
</ol>
+
<li>Mark the PCR tubes on lid and side and place in thermocycler.</li>
 +
<li>The annealing temperature should match the primer melting temperatures. This can be hard as Tm predictors tend to disagree.</li>
 +
<li>Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).</li>
 +
</ul>
</td>
</td>
</tr>
</tr>
 +
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h1>Glycerol stock-preservation</h1></th>
+
<th colspan="2" scope="col"><h1>Casting of gels and gel electrophoresis (remember to include EtBr)</h1></th>
</tr>     
</tr>     
<tr>
<tr>
Line 59: Line 63:
<ol>
<ol>
-
<li>Add one ml sterile 50% glycerol to a cryotube.</li>
+
<li>A</li>
-
<li>Add one ml overnight culture to a cryotube from step 1</li>
+
<li>A</li>
-
<li>Store at -80 °C</li>
+
<li>S</li>
</ol>
</ol>
Line 71: Line 75:
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h1>Glycerol stock-preservation</h1></th>
+
<th colspan="2" scope="col"><h1>Preparation of Cam stock solution</h1></th>
</tr>     
</tr>     
<tr>
<tr>
Line 79: Line 83:
<ol>
<ol>
-
<li>Add one ml sterile 50% glycerol to a cryotube.</li>
+
<li>h</li>
-
<li>Add one ml overnight culture to a cryotube from step 1</li>
+
<li>u</li>
-
<li>Store at -80 °C</li>
+
<li>i</li>
</ol>
</ol>
Line 91: Line 95:
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h1>Glycerol stock-preservation</h1></th>
+
<th colspan="2" scope="col"><h1>Recipe on M9 minimal media</h1></th>
</tr>     
</tr>     
<tr>
<tr>
Line 99: Line 103:
<ol>
<ol>
-
<li>Add one ml sterile 50% glycerol to a cryotube.</li>
+
<li>Make M9 salts</li>
-
<li>Add one ml overnight culture to a cryotube from step 1</li>
+
<li>To make M9 Salts aliquot 800ml H2O and add</li>
-
<li>Store at -80 °C</li>
+
        <ul>
 +
            <li>64g Na2HPO4-7H2O</li>
 +
            <li>15g KH2PO4</li>
 +
            <li>2.5g NaCl</li>
 +
            <li>5.0g NH4Cl</li>
 +
            <li>Stir until dissolved</li>
 +
            <li>Adjust to 1000ml with distilled H2O</li>
 +
            <li>Sterilize by autoclaving</li>
 +
                      </ul>
 +
<li>Measure ~700ml of distilled H2O (sterile)</li>
 +
<li>Add 200ml of M9 salts</li>
 +
<li>Add 2ml of 1M MgSO4 (sterile)</li>
 +
<li>Add 20 ml of 20% glucose (or other carbon source)</li>
 +
<li>Add 100ul of 1M CaCl2 (sterile)</li>
 +
<li>Adjust to 1000ml with distilled H2O</li>
 +
 
</ol>
</ol>
Line 111: Line 130:
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h1>Glycerol stock-preservation</h1></th>
+
<th colspan="2" scope="col"><h1>Restriction analysis</h1></th>
</tr>     
</tr>     
<tr>
<tr>
Line 119: Line 138:
<ol>
<ol>
-
<li>Add one ml sterile 50% glycerol to a cryotube.</li>
+
<li>Make a mastermix containing.</li>
-
<li>Add one ml overnight culture to a cryotube from step 1</li>
+
  <ul>
-
<li>Store at -80 °C</li>
+
            <li>1X NEBuffer (according to the desired restriction enzyme)</li>
 +
            <li>1X BSA (note if NEBuffer is called X.1 BSA is already added in the buffer)</li>
 +
            <li>1 unit/μl restriction enzyme</li>
 +
           
 +
                      </ul>
 +
<li>Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl</li>
 +
<li>Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes</li>
 +
<li>Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml</li>
 +
<li>Analyze your gel</li>
</ol>
</ol>
Line 127: Line 154:
</td>
</td>
</tr>
</tr>
 +
</table>
</table>
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h2>Rasmus Bech</h2></th>
+
<th colspan="2" scope="col"><h1>next</h1></th>
</tr>     
</tr>     
<tr>
<tr>
-
+
<td>
 +
  <ul>
 +
            <li></li>
 +
            <li></li>
 +
            <li></li>
 +
           
 +
                      </ul>
 +
<ol>
 +
<li></li>
 +
<li></li>
 +
<li></li>
 +
<li></li>
 +
</ol>
 +
 
-
<b>Motivation</b><br>
 
-
I joined iGEM in order to take part in a project where the students have more control and responsibility than other projects at DTU. It was also a motivation being part of a team, as large projects at DTU most often are individual.<br>
 
-
<br>
 
-
<b>Major contributions</b><br>
 
-
<br>
 
-
<b>How do you prefer spinach?</b><br>
 
-
I prefer spinach in a salad with strawberry, red onion, pine nuts, feta and a dressing with Balsamico vinegar, oil, sugar, soy sauce and tabasco.<br>
 
</td>
</td>
Line 150: Line 184:
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<table width="100%" border="0" cellspacing="15" cellpadding="0" style="background-color:transparent;">
<tr>
<tr>
-
<th colspan="2" scope="col"><h2>Thor Bech Johannesen</h2></th>
+
<th colspan="2" scope="col"><h1>Protocol for eating spinach</h1></th>
</tr>     
</tr>     
<tr>
<tr>
<td rowspan="1" scope="col" width="300px">
<td rowspan="1" scope="col" width="300px">
-
<img style="float: left; "src="https://static.igem.org/mediawiki/2014/2/23/Thor.jpg" height="300"> </img>
+
<img style="float: left; "src="http://i.perezhilton.com/wp-content/uploads/2010/03/popeye46704__oPt.jpg" height="300"> </img>
</td>
</td>
<td>
<td>
-
<b>Motivation</b><br>
+
 
-
As a student at DTU you get to do plenty of project work, however it is mostly carried out individually or in a small group, with very specific goals and a high degree of supervision. iGEM offers the opportunity to work on a much larger project with a high degree of autonomy, where everything from the overall goals and strategy to design of experiments is left for the students to manage. This provides a great opportunity to experience the challenges of actual research projects<br>
+
-
<br>
+
-
<b>Major contributions</b><br>
+
-
<br>
+
-
<br>
+
-
<b>How do you prefer spinach?</b><br>
+
-
Luminescent and far away from my taste buds.<br>
+
</td>
</td>
Line 177: Line 204:
</tr>     
</tr>     
<tr>
<tr>
-
<td rowspan="1" scope="col" width="300px">
+
-
 
+
-
<img style="float: left; "src="https://static.igem.org/mediawiki/2014/4/47/Anne_Sofie.jpg" height="300"> </img>
+
-
</td>
+
<td>
<td>

Latest revision as of 14:32, 1 August 2014

Protocols

Glycerol stock-preservation
  1. Add one ml sterile 50% glycerol to a cryotube.
  2. Add one ml overnight culture to a cryotube from step 1
  3. Store at -80 °C

PCR
  • Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
  • The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
  • Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
  • Mark the PCR tubes on lid and side and place in thermocycler.
  • The annealing temperature should match the primer melting temperatures. This can be hard as Tm predictors tend to disagree.
  • Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

Casting of gels and gel electrophoresis (remember to include EtBr)
  1. A
  2. A
  3. S

Preparation of Cam stock solution
  1. h
  2. u
  3. i

Recipe on M9 minimal media
  1. Make M9 salts
  2. To make M9 Salts aliquot 800ml H2O and add
    • 64g Na2HPO4-7H2O
    • 15g KH2PO4
    • 2.5g NaCl
    • 5.0g NH4Cl
    • Stir until dissolved
    • Adjust to 1000ml with distilled H2O
    • Sterilize by autoclaving
  3. Measure ~700ml of distilled H2O (sterile)
  4. Add 200ml of M9 salts
  5. Add 2ml of 1M MgSO4 (sterile)
  6. Add 20 ml of 20% glucose (or other carbon source)
  7. Add 100ul of 1M CaCl2 (sterile)
  8. Adjust to 1000ml with distilled H2O

Restriction analysis
  1. Make a mastermix containing.
    • 1X NEBuffer (according to the desired restriction enzyme)
    • 1X BSA (note if NEBuffer is called X.1 BSA is already added in the buffer)
    • 1 unit/μl restriction enzyme
  2. Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl
  3. Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes
  4. Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml
  5. Analyze your gel

next

Protocol for eating spinach

Overview

Retrieved from "http://2014.igem.org/Team:DTU-Denmarkgogo"