Seed DNA Extraction in 96-Deep Well Plates

DNA extraction procedure modified from: Pallotta M.A .et al. (2003) Marker assisted wheat breeding in the southern region of Australia. Proceedings of the Tenth International Wheat Genetics Symposium (1-6 September, 2003, Paestum, Italy) p.789-791. Patricia Warner patricia.warner@adelaide.edu.au Submitted by Shiaoman Chao chaos@fargo.ars.usda.gov and Daryl Somers somersd@agr.gc.ca

Sample preparation:

• Seed: For seed DNA, seeds are crushed with a hammer and approximately 1/3 of the seed is placed in a deep well plate. Avoid excess amounts of the seed coat.

• Tissue: The tissue is harvested and lyophilized in deep well plates and shaken on a paint shaker for with beads 1-2 mins to grind the tissue to a fine powder.

Extraction:

The scale can be modified to fit small 96 well plates or individual tubes as desired, adjust volumes accordingly. The protocol as written is for deep 96 well plates.

1. Preheat extraction buffer to 65°C allow the plates containing the tissue to warm up to room temperature if they have been stored at -20°C. (plates can be stored at room temp)

2. Extraction Buffer (0.1M Tris-HCl pH 7.5, 0.05 EDTA pH 8.0, 1.25% SDS).
   For 1 liter:

   • 100 ml 1.0 M Tris-HCl pH 7.5

   • 100 ml 0.5M EDTA pH 8.0

   • 125 ml 10% SDS

   • 675 ml ddH₂O

3. Add 500µl of extraction buffer to each well, seal the plates with lids or use clear plastic seals to seal plates and mix. (Gently shake deep well plates, tubes can be more thoroughly mixed.)

4. Incubate the plate 65°C 45min-1hour.

5. Place the plates at 4°C to cool them down to room temperature (about 15 minutes) before adding 250µl 5M NaCl, which is stored at 4°C. Pipette mix in plates (or shake thoroughly in tubes) to mix in the NaCl and then leave to stand for 15 minutes at 4°C (or 10 minutes at -20°C).

6. Centrifuge the plate for 15 minutes at 3800 rpm to collect the precipitated proteins and plant tissue.

7. Recover about 600µl of the supernatant into new sterile 96 deep-well plates or (micro tubes) containing 360µl of iso-propanol in each well. Mix thoroughly and allow the DNA to precipitate for about 10 minutes -20°C.

8. Centrifuge the samples for 20 minutes at 3800 rpm. Allow the remaining fluid to drain off the DNA pellet by inverting the tubes onto a piece of paper towel. ONLY INVERT THE TUBES FOR LESS THAN 1 MINUTE OTHERWISE YOU WILL LOSE THE DNA PELLETS.

9. Wash the pellet in 500µl of 70% ethanol.

10. Centrifuge the plate for 15 minutes at 3800 rpm and again discard the supernatant.

11. Resuspend the pellet in 100µl of 1X TE or (PCR grade water) for seed DNA or 200µl for leaf DNA. Leave the DNA to dissolve overnight at 4°C in the fridge. Try to dislodge the pellet.

12. Quantify DNA if necessary.