Griffitts:Plant DNA preparation

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)
• T₃E_0.3
• 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 T₃E_0.3
  • DON'T pipette up and down!
• Heat at 65°C for 5 minutes with flicking

Optional:

• Run 5 μL of each sample on an electrophoresis gel