Add 500μl of extraction buffer for a total volume of 600μl. Previously, 100μl (refer to step 8) was added to aid in resuspending the cell powder after LN grinding.
Incubate the samples at 55-60°C for 1 hour.
Add 700μl of Chloroform:Phenol:Isoamyl alcohol and mix well to form an emulsion by pipetting up and down, shaking the tubes, and/or vortexing. In the case of extra dirty samples, they can be placed in a rack on an orbital shaker for 10-30 minutes to increase the contact of the two liquid phases. This will help further remove debris and pigments from the samples.
Centrifuge for 10 minutes at maximum speed (13-15,000 rpm). After centrifugation, you should have three layers: top - aqueous phase, middle - debris and proteins, bottom - chloroform. Proceed to the next step quickly to avoid remixing of the phases.
Pipette off the aqueous phase (top), taking care not to suck up any of the middle or chloroform phases.
Transfer the aqueous phase into a new labeled 1.5 ml tube.
Add 4μl of RNAse A (Quiagen or prepared from powder 1mg/ml - keep aliquots frozen until use. Do not re-freeze aliquots multiple times) and mix by inverting. Incubate at 37°C for 30 minutes.
Repeat steps 8-11 (second time, centrifuge for 5 minutes).
Estimate the volume of the aqueous phase, which should be approximately 450μl.
Add 0.1 volume of cold sodium Acetate 3M (final concentration 0.3M). For example, for 450μl, add 45μl.
Add 0.7-0.9 volumes (using the combined volume of aqueous phase and added NaAc) of cold molecular-grade isopropanol. This should be approximately 400μl for 500μl CTAB. Mix well.
Let it sit in the freezer for 45 minutes to an hour. Longer times (i.e., overnight) will tend to yield more DNA but also more contaminants.
Centrifuge for 5 minutes at maximum speed. Orient the tubes equally to facilitate subsequent removal of supernatant without disturbing the DNA pellet.
Pour or pipette off the liquid, being careful not to lose the DNA pellet, which is very loose and difficult to see at this stage.
Add 700μl of cold 70% Ethanol and invert once to mix.
Centrifuge for 1 minute at maximum speed.
Pipette off the liquid, being careful not to lose the DNA pellet.
Repeat steps 18-20.
Pour or pipette off the liquid, being careful not to lose the DNA pellet. If you can't remove all the ethanol, that is okay. It is better to leave some ethanol than risk sucking up your DNA!
Dry the pellet by inverting the tubes on a dry, clean paper towel.
Resuspend the samples with 45μl of TE. Allow them to resuspend for 1 hour at room temperature or overnight at 4°C before using. (I favor overnight resuspension. Usually, it won't require much pipetting to resuspend the pellet if it has been rehydrating overnight).