DNA Isolation
- Perform in a fume hood with protective goggles.
- Add 300-400 mg dry tissue to a 15mL Falcon Tube (screw cap).
- Add 7 mL CTAB stock solution (with freshly added β-mercaptoethanol). Vortex to mix well.
- Incubate 90 min. at 65°C. Invert every 15 minutes.
- Remove tubes from the bath and cool 10 min.
- Add 8mL 24:1 Chloroform: IsoAmyl alcohol.
- Invert for 10 min. on the rocker.
- Centrifuge for 10 min. at 3,700 rpm.
- Pour off the supernatant into a new snap-top tube.
- Add 5uL of 20 mg/mL Rnase A. Incubate 30 min. at 37°C.
- Add 4 ml C/I and shake 10 min.
- Centrifuge for 10 min. at 3,700 rpm.
- Remove the supernatant with transfer pipette into a new snap-top tube.
- Add 5 ml isopropanol to precipitate DNA.
- Remove DNA with glass hook or spin down to pellet the precipitated DNA
- Re-suspend DNA in 300-500ul nuclease-free water.
- Transfer to a 1.5mL tube.
- Quantify DNA using a nanodrop.
DNA Cleanup
- Add equal volume of phenol:chloroform to DNA sample and mix well/vortex gently.
- Spin at 8,000rpm for 3 minutes.
- Transfer the aqueous layer to a new 1.5mL tube and add equal volume chloroform.
- Spin at 8,000rpm for 3 minutes.
- Transfer the aqueous layer to a new 1.5mL tube.
- Add 0.1 volume cold 3M Na-Acetate (pH 5.5) and 2 volumes cold 100% ethanol. Incubate for 30 minutes to 1 hour at -20°C.
- Spin at 10,000rpm for 10 minutes, 4°C.
- Remove supernatant and wash with 400uL cold 70% ethanol.
- Spin at 10,000rpm for 5 minutes and remove ethanol.
- Air dry for 5-10 minutes.
- Resuspend DNA in 200uL nuclease-free water.
- Quantify DNA using a nanodrop.