Griffitts:Plant DNA preparation
From OpenWetWare
Jump to navigationJump to search
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:
- Run 5 μL of each sample on an electrophoresis gel