Large Scale Preps

1. Turn on water bath to 65 degree.
2. Add 300-400 mg dry tissue to a 15 ml Falcon Tube.
3. Add 7 ml CTAB stock solution (making sure to add B-mercaptoethanol). Vortex to mix well.
4. Incubate 90 min. at 65 degrees C. Invert every 15 minutes
5. Remove tubes from the bath and cool 10 min. Add 8 ml 24:1 Chloroform: IsoAmyl alcohol (C/I) in fume hood. Invert for 10 min. on the rocker.
6. Spin in centrifuge for 10 min. at 3700 rpm. You may need to take off the top rack of the bucket.
7. Pour off the supernatant into a new snap-top tube.
8. Add 50 ulof 25 mg/ml (or 125 ul of 10 mg/ml) Rnase A (from freezer). Incubate 30 min. at 37 degrees C.
9. Add 4 ml C/I and shake 10 min. Make sure to close the snap top very tightly!
10. Spin 10 min. at 3700 rpm. Remove the supernatant with transfer pipette into a new snap-top tube.
11. Add 5 ml isopropanol to precipitate DNA.
12. Remove DNA with glass hook.
13. Re-suspend DNA in 300ul TE.

Two Tube Small Scale Prep

1. Grind Tissue
2. Add Tissue to 700ul CTAB, vortex
3. Incubate at 65C for 60-90 minutes, invert every 15 min.
4. Add 400ul Chloroform:IAA, vortex
5. Spin 5 minutes
6. Transfer top phase, Add 300ul Isopropanol. Mix by inversion
7. Spin 5 minutes.
8. Pour off top phase.
9. Add 500ul 70% EtOH, vortex.
10. Spin 5 minutes.
11. Pour off top phase, dry.
12. Add 250ul low Tris (ph8)

Three Tube Small Scale Prep

1. Grind Tissue
2. Add Tissue to 700ul CTAB, vortex
3. Incubate at 65C for 60-90 minutes, invert every 15 min.
4. Add 400ul Chloroform:IAA, vortex
5. Spin 5 minutes
6. Transfer top phase to new tube
7. Add 400ul Chloroform:IAA, vortex
8. Spin 5 minutes.
9. Transfer top phase, Add 300ul Isopropanol. Mix by inversion
10. Spin 5 minutes.
11. Pour off top phase.
12. Add 500ul 70% EtOH, vortex.
13. Spin 5 minutes.
14. Pour off top phase, dry.
15. Add 250ul low Tris (ph8)