Rapid DNA Extraction Protocol
Submitted by Jim Anderson
DNA extraction procedure
- Harvest an approximately 2 cm sized leaf from a seedling (1-2 leaf stage) into a 1.5
mL tube and keeps them in liquid nitrogen.
- Grind leaf in the 1.5 mL tube using a cold glass rod (kept in liquid nitrogen) over the vortex.
- Extract DNA immediately or store samples in -80蚓
| Buffer, total volume = 50 mL.
- Heat solution 30 sec. in 1000W microwave (to 65蚓).
- Place on heat plate to maintain temp.
- Dispense 700 無 warm buffer into tube, invert to mix thoroughly.
- Incubate in water bath at 65蚓 for 30 min.
- Invert every 5 min.
- Dispense 700 無 24:1 Chloroform/Isoamylic alcohol into tube, mix vigorously.
- Spin in microfuge 10 min @10,000 rpm.
- Carefully remove from tubes and keep at slant in a rack.
- Pipette about 500 無 of aqueous layer from the top of the tube. Transfer to new tube.
- Add 1 mL of cold 95% ethanol, invert to precipitate the DNA.
- Spin DNA to bottom of tube (10 minute at 10,000 rpm).
- Decant, and wash pellet in 1 mL of 70% ethanol.
- Spin the tubes (1 min, 10,000 rpm) and pour out 70% ethanol and blot tube on Kimwipe.
- Incubate at 50蚓 for 30 min. to dry pellets.
- Resuspend in 150 無 TE buffer.
- Dissolve in water bath at 65蚓 for about 6 hrs.
- Prepare 1:5 dilutions for PCR.