Difference between revisions of "Team:DTU-Denmark/Experiments"

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<div class="col-xs-12">
 
<div class="col-xs-12">
  
−
<p style="font-size: 70%;" >
+
<p>
 
DNA from IDT will typically be delivered in a white flaky substance, which need to be resuspended, before the DNA is ready for use
 
DNA from IDT will typically be delivered in a white flaky substance, which need to be resuspended, before the DNA is ready for use
 
</p>
 
</p>
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<ul>
 
<ul>
−
   <li><p style="font-size:90%">Table centrifuge</p></li>
+
   <li><p>Table centrifuge</p></li>
−
  <li><p style="font-size:90%">EB/TE buffer</p></li>
+
  <li><p>EB/TE buffer</p></li>
−
<li><p style="font-size:90%">Genes/Primers from IDT or other DNA provider</p></li>
+
<li><p>Genes/Primers from IDT or other DNA provider</p></li>
 
</ul>  
 
</ul>  
  
Line 60: Line 60:
  
 
<ol>
 
<ol>
−
   <li><p style="font-size:90%">Quickly spin the DNA down in the table centrifuge</p></li>
+
   <li><p>Quickly spin the DNA down in the table centrifuge</p></li>
 
   
 
   
−
<li><p style="font-size:90%">Calculate the amount of EB buffer need to dilute the gblocks to a desired concentration. Important: gene fragments and primeres are not diluted to the same concentration: The concentration of gene fragments is usually 25 ng/μL  and for primers it is 100 μM.</p></li>
+
<li><p>Calculate the amount of EB buffer need to dilute the gblocks to a desired concentration. Important: gene fragments and primeres are not diluted to the same concentration: The concentration of gene fragments is usually 25 ng/μL  and for primers it is 100 μM.</p></li>
  
−
<li><p style="font-size:90%">Genes/Primers from IDT or other DNA provider</p>
+
<li><p>Genes/Primers from IDT or other DNA provider</p>
 
<ul>
 
<ul>
−
   <li><p style="font-size:90%">DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is  40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples)</p></li>
+
   <li><p>DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is  40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples)</p></li>
  
−
  <li><p style="font-size:90%"> In order to calculate the the molar amount of primer, use the <a href="https://nebiocalculator.neb.com/#!/ssdnaamt">NEB calculator</a></p></li>
+
  <li><p> In order to calculate the the molar amount of primer, use the <a href="https://nebiocalculator.neb.com/#!/ssdnaamt">NEB calculator</a></p></li>
 
</ul>  
 
</ul>  
 
</li>
 
</li>
  
−
<li><p style="font-size:90%">Add the calculated amount of EB buffer</p></li>
+
<li><p>Add the calculated amount of EB buffer</p></li>
  
−
<li><p style="font-size:90%">Store the resuspended DNA at -20°C </p></li>
+
<li><p>Store the resuspended DNA at -20°C </p></li>
 
</ol>  
 
</ol>  
  
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−
<p style="font-size: 70%;" >
+
<p>
 
Basic protocol on plating of the fungi.  
 
Basic protocol on plating of the fungi.  
 
</p>
 
</p>
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<ul>
 
<ul>
−
   <li><p style="font-size:90%">Agar plates with media of interest</p></li>
+
   <li><p>Agar plates with media of interest</p></li>
−
  <li><p style="font-size:90%">Sterile toothpicks</p></li>
+
  <li><p>Sterile toothpicks</p></li>
 
</ul>  
 
</ul>  
  
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<h4 class="media heading">Procedure</h4>
 
<h4 class="media heading">Procedure</h4>
−
<p style="font-size:90%">Plating using mycelia
+
<p>Plating using mycelia
 
</p>
 
</p>
 
<ol>
 
<ol>
−
   <li><p style="font-size:90%">Open the plate containing the mycelia from the species of interest</p></li>
+
   <li><p>Open the plate containing the mycelia from the species of interest</p></li>
 
   
 
   
−
<li><p style="font-size:90%">With a sterile toothpick, scratch the surface of the mycelia.</p></li>
+
<li><p>With a sterile toothpick, scratch the surface of the mycelia.</p></li>
  
−
<li><p style="font-size:90%">Spike the new agar plate with the toothpick.</p>
+
<li><p>Spike the new agar plate with the toothpick.</p>
 
</li>
 
</li>
  
−
<li><p style="font-size:90%">Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle. </p></li>
+
<li><p>Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle. </p></li>
  
−
<li><p style="font-size:90%">Put the plate into a plastic growth bag. </p></li>
+
<li><p>Put the plate into a plastic growth bag. </p></li>
  
−
<li><p style="font-size:90%">Place growing bag into the incubator.  </p></li>
+
<li><p>Place growing bag into the incubator.  </p></li>
 
</ol>  
 
</ol>  
  
−
<p style="font-size:90%">Plating using spores
+
<p>Plating using spores
 
</p>
 
</p>
 
<ol>
 
<ol>
−
   <li><p style="font-size:90%">Touch the sterile tooth pick in the spore suspension.</p></li>
+
   <li><p>Touch the sterile tooth pick in the spore suspension.</p></li>
 
   
 
   
−
<li><p style="font-size:90%">Spike the new agar plate with the toothpick.</p></li>
+
<li><p>Spike the new agar plate with the toothpick.</p></li>
  
−
<li><p style="font-size:90%">Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle. </p>
+
<li><p>Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle. </p>
 
</li>
 
</li>
  
−
<li><p style="font-size:90%">Put the plate into a plastic growth bag.  </p></li>
+
<li><p>Put the plate into a plastic growth bag.  </p></li>
  
−
<li><p style="font-size:90%">Place growing bag into the incubator. </p></li>
+
<li><p>Place growing bag into the incubator. </p></li>
  
 
</ol>  
 
</ol>  
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<div class="row">
 
<div class="row">
 
<div class="col-xs-12">
 
<div class="col-xs-12">
−
<p style="font-size: 70%;" >
+
<p>
 
Minimal media used for protoplastation of A. oryzae. This is for 1 L of media.  
 
Minimal media used for protoplastation of A. oryzae. This is for 1 L of media.  
 
</p>
 
</p>
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<ul>
 
<ul>
−
   <li><p style="font-size:90%">50 mL D-glucose </p></li>
+
   <li><p>50 mL D-glucose </p></li>
−
  <li><p style="font-size:90%">50 mL Nitrate salts</p></li>
+
  <li><p>50 mL Nitrate salts</p></li>
−
<li><p style="font-size:90%">1 mL Trace elements</p></li>
+
<li><p>1 mL Trace elements</p></li>
−
  <li><p style="font-size:90%">1 mL Thiamine</p></li>
+
  <li><p>1 mL Thiamine</p></li>
−
<li><p style="font-size:90%">20 g Agar (SO.BI.GEL) </p></li>
+
<li><p>20 g Agar (SO.BI.GEL) </p></li>
 
</ul>  
 
</ul>  
  
Line 220: Line 220:
  
 
<h4 class="media heading">Procedure</h4>
 
<h4 class="media heading">Procedure</h4>
−
<p style="font-size:90%">Mix
+
<p>Mix
 
</p>
 
</p>
 
<ol>
 
<ol>
−
   <li><p style="font-size:90%">Mix in a blue cap flask.</p></li>
+
   <li><p>Mix in a blue cap flask.</p></li>
 
   
 
   
−
<li><p style="font-size:90%">Add water until the volume is 1 L. </p></li>
+
<li><p>Add water until the volume is 1 L. </p></li>
  
−
<li><p style="font-size:90%">Autoclave. </p>
+
<li><p>Autoclave. </p>
 
</li>
 
</li>
  

Revision as of 15:54, 4 October 2018

Experiments

Synthlab protocols

DNA from IDT will typically be delivered in a white flaky substance, which need to be resuspended, before the DNA is ready for use

Materials

  • Table centrifuge

  • EB/TE buffer

  • Genes/Primers from IDT or other DNA provider

Procedure

  1. Quickly spin the DNA down in the table centrifuge

  2. Calculate the amount of EB buffer need to dilute the gblocks to a desired concentration. Important: gene fragments and primeres are not diluted to the same concentration: The concentration of gene fragments is usually 25 ng/μL and for primers it is 100 μM.

  3. Genes/Primers from IDT or other DNA provider

    • DNA from IDT usually comes in dried flakes of 500 or 1000 ng of DNA. To achieve the desired concentration (usually 25 ng/μL) the needed amount of EB buffer is 40 μL (for 1000 ng samples) or 20 μL (for 500 ng samples)

    • In order to calculate the the molar amount of primer, use the NEB calculator

  4. Add the calculated amount of EB buffer

  5. Store the resuspended DNA at -20°C

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.

Mycolab protocols

Basic protocol on plating of the fungi.

Materials

  • Agar plates with media of interest

  • Sterile toothpicks

Procedure

Plating using mycelia

  1. Open the plate containing the mycelia from the species of interest

  2. With a sterile toothpick, scratch the surface of the mycelia.

  3. Spike the new agar plate with the toothpick.

  4. Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.

  5. Put the plate into a plastic growth bag.

  6. Place growing bag into the incubator.

Plating using spores

  1. Touch the sterile tooth pick in the spore suspension.

  2. Spike the new agar plate with the toothpick.

  3. Repeat steps 1-3 times. Three inoculation points should be made in the agar making forming a triangle.

  4. Put the plate into a plastic growth bag.

  5. Place growing bag into the incubator.

Minimal media used for protoplastation of A. oryzae. This is for 1 L of media.

Materials

  • 50 mL D-glucose

  • 50 mL Nitrate salts

  • 1 mL Trace elements

  • 1 mL Thiamine

  • 20 g Agar (SO.BI.GEL)

Procedure

Mix

  1. Mix in a blue cap flask.

  2. Add water until the volume is 1 L.

  3. Autoclave.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.