Marker Assisted Selection in Wheat - HOME USDA National Institute of Food and Agriculture The Borlaug Global Rust Initiative Marker Assisted Selection in Wheat
Home
Back to protocols
News for members 

Rapid DNA Extraction Protocol

Submitted by Jim Anderson

DNA extraction procedure

Sample preparation:

  1. 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.
  2. Grind leaf in the 1.5 mL tube using a cold glass rod (kept in liquid nitrogen) over the vortex.
  3. Extract DNA immediately or store samples in -80蚓

Extraction:

Buffer, total volume = 50 mL. 
Component Concentration Volume, mL
dd H2O - 32.0
EDTA 0.5 M 5.0
Tris HCL 1.0 M 5.0
NaCl 5.0 M 5.0
SDS 20% 3.1
NaOH 10 M 152 無
Na-bisulfite 0.19 g -
  1. Heat solution 30 sec. in 1000W microwave (to 65蚓).
  2. Place on heat plate to maintain temp.
  3. Dispense 700 無 warm buffer into tube, invert to mix thoroughly.
  4. Incubate in water bath at 65蚓 for 30 min.
  5. Invert every 5 min.
  6. Dispense 700 無 24:1 Chloroform/Isoamylic alcohol into tube, mix vigorously.
  7. Spin in microfuge 10 min @10,000 rpm.
  8. Carefully remove from tubes and keep at slant in a rack.
  9. Pipette about 500 無 of aqueous layer from the top of the tube. Transfer to new tube.
  10. Add 1 mL of cold 95% ethanol, invert to precipitate the DNA.
  11. Spin DNA to bottom of tube (10 minute at 10,000 rpm).
  12. Decant, and wash pellet in 1 mL of 70% ethanol.
  13. Spin the tubes (1 min, 10,000 rpm) and pour out 70% ethanol and blot tube on Kimwipe.
  14. Incubate at 50蚓 for 30 min. to dry pellets.
  15. Resuspend in 150 無 TE buffer.
  16. Dissolve in water bath at 65蚓 for about 6 hrs.
  17. Prepare 1:5 dilutions for PCR.
[Back to top]