Griffitts:Plant DNA preparation

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Revision as of 13:50, 12 October 2010 by Matthew B. Crook (Talk | contribs)
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Materials

  • Plant material
  • Liquid nitrogen
  • Pestles
  • Ice
  • 70% ethanol
  • Qiagen Cell Lysis Solution (on shelf with Qiagen kits)
  • Qiagen Protein Precipitation Solution (on shelf with Qiagen kits)
  • T3E0.3
  • 1.7-mL microcentrifuge tubes

Procedure

  • Cut 1 leaf from plant and place in a microcentrifuge tube
  • Dip in liquid nitrogen for 1 second, pour off excess
  • Right as the liquid nitrogen boils off, add 500 μL Qiagen Cell Lysis Solution
  • Grind with pestle
  • Vortex 5 seconds
  • Heat at 65°C for 1 hour (vortex occasionally)
  • Chill to room temperature on ice
    • This takes about 4 minutes
  • Add 180 μL of Qiagen Protein Precipitation Solution
  • Vortex for 20 seconds
  • Incubate on ice for 15 minutes
  • Centrifuge at maximum speed for 6 minutes
  • Move 500 μL of the supernatant to a new tube
  • Add 500 μL isopropanol
  • Invert 3 times
  • Wait 10 minutes
  • Centrifuge at maximum speed for 6 minutes
  • Pour off supernatant
  • Resuspend in 70% ethanol
  • Vortex for 3 seconds
  • Centrifuge at maximum speed for 6 minutes
  • Pour off supernatant
  • Centrifuge at maximum speed for 30 seconds
  • Remove remaining supernatant using a P-200
  • Add 80 μL T3E0.3
    • DON'T pipette up and down!
  • Heat at 65°C for 5 minutes with flicking

Optional:

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