Difference between revisions of "Team:WashU StLouis/Experiments"

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   <ul>
 
   <ul>
−
<li> step 1 </li>
+
<li>Centrifuge (X mL) cell culture for 15 minutes at 5000g</li>
−
<li> step 2 </li>
+
<li>Discard supernatant and resuspend in 1 mL of cloning water</li>
−
<li> step 3 </li>
+
<li>Transfer solution into bashing bead lysis/filtration tube</li>
 +
<li>Add 6 mL of fungal/bacterial DNA binding buffer</li>
 +
<li>Vortex for 5 minutes</li>
 +
<li>Spin down for 5 minutes at 5000g</li>
 +
<li>Transfer filter to a 50 mL tube</li>
 +
<li>Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g</li>
 +
<li>Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once</li>
 +
<li>Add 200 uL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times</li>
 +
<li>Transfer column to a 1.5 mL microcentrifuge tube and add 30 uL of cloning water</li>
 +
<li>Let sit for 4 minutes, then spin for 4 minutes at 10,000 g</li>
 +
<li>Measure concentration of gDNA and label tube</li>
 +
</ul>
 +
 
 
</div>
 
</div>
  
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<div class="protocol-panel">
 
<div class="protocol-panel">
 
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−
  <ul>
+
<li>Add the following to a single PCR tube:</li>
−
<li> step 1 </li>
+
<ul>
−
<li> step 2 </li>
+
<li>20-21.5 uL Cloning Water (depending on type of amplicon)</li>
−
<li> step 3 </li>
+
<li>10 uL of Betaine</li>
 +
<li>10 uL of 5x High-Fidelity Buffer </li>
 +
<li>4 uL DMSO</li>
 +
<li>2.5 uL 10 uM combined forward/reverse primers</li>
 +
<li>1 uL DNTPs</li>
 +
<li>0.5 uL of plasmid or 2 uL of gDNA</li>
 +
<li>0.5 uL of Phusion Polymerase (kept on ice)</li>
 +
</ul>
 +
<li>Immediately run in a thermocycler for the following times/temperatures</li>
 +
<ul>
 +
<li>Initial Denaturation 98C 30 sec</li>
 +
<li>25-35 cycles</li>
 +
<ul>
 +
<li>98 C 5-10 sec</li>
 +
<li>45-72 C (at annealing temp) 10-30 sec</li>
 +
<li>72 C 15-30 sec/(length of amplicon in kb)</li>
 +
</ul>
 +
<li>Final Extension 72 C 5-10 minutes</li>
 +
<li>Hold at 4C</li>
 +
</ul>
 +
<li>Store at 4C until use</li>
 +
</ul>
 +
 
 
</div>
 
</div>
  

Revision as of 22:04, 15 October 2016

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Protocols

Check out the protocols we used for our project



Cloning

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  • Centrifuge (X mL) cell culture for 15 minutes at 5000g
  • Discard supernatant and resuspend in 1 mL of cloning water
  • Transfer solution into bashing bead lysis/filtration tube
  • Add 6 mL of fungal/bacterial DNA binding buffer
  • Vortex for 5 minutes
  • Spin down for 5 minutes at 5000g
  • Transfer filter to a 50 mL tube
  • Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
  • Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
  • Add 200 uL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
  • Transfer column to a 1.5 mL microcentrifuge tube and add 30 uL of cloning water
  • Let sit for 4 minutes, then spin for 4 minutes at 10,000 g
  • Measure concentration of gDNA and label tube

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.

  • Add the following to a single PCR tube:
    • 20-21.5 uL Cloning Water (depending on type of amplicon)
    • 10 uL of Betaine
    • 10 uL of 5x High-Fidelity Buffer
    • 4 uL DMSO
    • 2.5 uL 10 uM combined forward/reverse primers
    • 1 uL DNTPs
    • 0.5 uL of plasmid or 2 uL of gDNA
    • 0.5 uL of Phusion Polymerase (kept on ice)
  • Immediately run in a thermocycler for the following times/temperatures
    • Initial Denaturation 98C 30 sec
    • 25-35 cycles
      • 98 C 5-10 sec
      • 45-72 C (at annealing temp) 10-30 sec
      • 72 C 15-30 sec/(length of amplicon in kb)
    • Final Extension 72 C 5-10 minutes
    • Hold at 4C
  • Store at 4C until use

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    • step 1
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    • step 1
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    BioBrick

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    • step 1
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    Measurement and Assays

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    Miscellaneous

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