Team:Bielefeld-Germany/Protocols/Overview

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<h1 align=center> This is how we work</h1>  
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<h1 align=center> This page lists all molecular genetics protocols we used in our project. </h1>  
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<p><font size=4 align=justify>
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This is an example
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Complete genome isolation from yeast
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with the Promega Wizard genomic DNA purification system kit
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<ul>
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  <li>Pellet 10 mL of over-night liquid culture grown in YPD broth in a 1.5 mL tube by centrifugation at 14,000 x g for 2 minutes.</li>
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  <li>Remove the supernatant.</li>
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  <li>Resuspend the cells in 90 μL of 50 mM EDTA.</li>
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  <li>Add 10 μL of 1000u lyticase and pipet 4 times to mix.</li>
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  <li>Incubate the sample at 37°C for 60 minutes to digest the cell wall</li>
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  <li>Centrifuge the sample at 14,000 × g for 2 minutes and then remove the supernatant.</li>
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  <li>Add 300 μl of Nuclei Lysis Solution to the cell pellet and pipet to mix.</li>
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  <li>Add 100 μl of Protein Precipitation Solution and vortex at high speed for 20 seconds.</li>
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  <li>Let the sample sit on ice for 5 minutes.</li>
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  <li>Centrifuge at 14,000 × g for 3 minutes.</li>
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  <li>Transfer the supernatant containing the DNA to a clean 1.5 ml tube containing 300 μl of room temperature isopropanol.</li>
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  <li>Gently mix by inversion until the DNA is visible.</li>
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  <li>Centrifuge at 14,000 × g for 2 minutes.</li>
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  <li>Carefully decant the supernatant and drain the tube on clean absorbent paper.</li>
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  <li>Add 300 μl of room temperature 70% ethanol and invert the tube several times to wash the DNA pellet.</li>
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  <li>Centrifuge at 14,000 × g for 2 minutes.</li>
 +
  <li>Drain the tube on clean absorbent paper and allow the pellet to air-dry for 15 minutes.</li>
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  <li>Add 50 μl of DNA Rehydration Solution.</li>
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  <li>Add 1.5μl of RNase Solution to the purified DNA sample. Vortex the sample for 1 second and incubate at 37°C for 15 minutes.</li>
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  <li>Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube.</li>
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  <li>Store the DNA at 2–8°C.</li>
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</ul>
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Revision as of 21:15, 22 July 2012

This page lists all molecular genetics protocols we used in our project.

Complete genome isolation from yeast with the Promega Wizard genomic DNA purification system kit

  • Pellet 10 mL of over-night liquid culture grown in YPD broth in a 1.5 mL tube by centrifugation at 14,000 x g for 2 minutes.
  • Remove the supernatant.
  • Resuspend the cells in 90 μL of 50 mM EDTA.
  • Add 10 μL of 1000u lyticase and pipet 4 times to mix.
  • Incubate the sample at 37°C for 60 minutes to digest the cell wall
  • Centrifuge the sample at 14,000 × g for 2 minutes and then remove the supernatant.
  • Add 300 μl of Nuclei Lysis Solution to the cell pellet and pipet to mix.
  • Add 100 μl of Protein Precipitation Solution and vortex at high speed for 20 seconds.
  • Let the sample sit on ice for 5 minutes.
  • Centrifuge at 14,000 × g for 3 minutes.
  • Transfer the supernatant containing the DNA to a clean 1.5 ml tube containing 300 μl of room temperature isopropanol.
  • Gently mix by inversion until the DNA is visible.
  • Centrifuge at 14,000 × g for 2 minutes.
  • Carefully decant the supernatant and drain the tube on clean absorbent paper.
  • Add 300 μl of room temperature 70% ethanol and invert the tube several times to wash the DNA pellet.
  • Centrifuge at 14,000 × g for 2 minutes.
  • Drain the tube on clean absorbent paper and allow the pellet to air-dry for 15 minutes.
  • Add 50 μl of DNA Rehydration Solution.
  • Add 1.5μl of RNase Solution to the purified DNA sample. Vortex the sample for 1 second and incubate at 37°C for 15 minutes.
  • Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube.
  • Store the DNA at 2–8°C.

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