CTAB DNA Extraction Protocol (3/29/00) Reagent CTAB ddH2O/Easypure 1M Tris 5M NaCl 0.5M EDTA -mercaptoethanol Volume 12.5mg 360L 62.5L 175L 25L 1.25L (single=630L) # of Samples Total 20 samples 250mg 7.20 mL 1.25mL 3.50mL 500L 25L 12.5mL 40 samples 500mg 14.40 mL 2.5mL 7mL 1mL 50L 25mL Total 1. 2. 3. 4. 5. 6. 7. 8. 9. Combine CTAB and water in sterile bottle. Swirl under hot tap water until CTAB has dissolved. Add other reagents in order. Move to hood to add the -mercaptoethanol. Add 50L mix to each sample in 1.5mL tube. Grind each sample separately with blue micropestle, leaving pestle in tube. Rinse the pestle with 550L mix into the tube and cap the tube. Place in 65C for 40-60 minutes. Label 2 sets of tubes. Centrifuge for 7 minutes @ 13K. Transfer upper phase into new 1.5mL tube. Do not transfer any solid material to new tube. Add equal volume (~550L) chloroform to each tube and finger vortex. Solution should be cloudy. 10. Centrifuge for 15 minutes @13K. 11. Transfer upper phase into new 1.5mL tube containing 750L cold isopropanol (found in freezer). Do not transfer any chloroform into new tube. 12. Place tubes in freezer for 30 minutes to overnight. 13. Centrifuge for 30 minutes @13K. 14. Decant supernatent into beaker. 15. Add 200L cold 70% ethanol (in freezer) to wash DNA pellet. 16. Centrifuge for 5 minutes @ 7K. 17. Decant supernatent into beaker and invert uncapped tubes on Kimwipes for 15-30 minutes, until dry. 18. Resuspend DNA pellet in 50L TE (100L for sporocarp samples). >If doing PCR same day, leave tubes at room temperature for 1 hour. >Otherwise, store DNA solutions at 4C.