DNA Precipitation of Restriction Digests

This protocol is used most often to prepare DNA for biolistic transformation as it removes protein/enzyme contaminants from restriction digestion reactions.

Materials:

 * 3 M NaOAc (pH 5.2)
 * Phenol:Choloroform (1:1 solution)
 * 100% cold or chilled EtOH
 * 70% EtOH room temperature
 * Restriction Digestion reaction
 * ddH2O or TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5)

Method:
''Step 2 should be performed under a chemical hood. Wear safety goggles and gloves. Phenol is caustic and can cause serious chemical burns. It is rapidly absorbed through the skin and inhaling vapors may have anesthetic effects and may prove fatal. Exercise extreme caution when working with chloroform too.''
 * 1) Add 30 uL of 3 M NaOAc to the digestion reaction and then add water to a total of 300 uL. Addition of 3 M NaOAc is 1/10 of total volume. For example, if the total volume is 500 you will add 50 uL of NaOAc. In this protocol, the total volume is 300 uL after addition of water, hence 30 uL of NaOAc.
 * 2) Add equal volume of phenol:choloroform solution and invert tubes 4-6 times. Centrifuge at 12,000 rpm for 10 min at RT. Carefully draw out top layer and transfer to new microcentrifuge tubes.Aqueous_Layer.png
 * 3) Add 1 mL of chilled 100% EtOH and place tubes at -20 C for 10 min.
 * 4) Centrifuge tubes at 12,000 rpm for 10 min at 4 C. Decant the supernatant immediately.
 * 5) Add 1 mL room temperature 70% EtOH and centrifuge again at 12,000 rpm for 30 seconds at 4 C. Pipette out the supernatant carefully.
 * 6) Place the tubes in a vacuum desiccator or air-dry for 5-10 min.
 * 7) Resuspend the precipitated DNA in 10 uL (or desired volume) of ddH2O or TE. DNA is more stable in TE.
 * 8) Store DNA in -20 C freezer.
 * 9) Analyze on agarose gel to ensure successful DNA precipitation.