Biology Reference
In-Depth Information
Table 5.1: Rapid Phenol Extraction of Genomic DNA from
D. melanogaster.
1. Homogenize 50-200 flies (frozen in liquid nitrogen) in a 15-ml polypropylene tube with a Teflon pestle in
2 ml of lysis buffer.
(Lysis buffer contains 100 mM Tris-HCl [pH 8.0], 50 mM NaCl, 50 mM EDTA, 1% SDS, 0.15 mM
spermine, and 0.5 mM spermidine.)
2. Add 20
μ
l of proteinase K solution (10 mg/ml).
3. Leave at 37°C for 1-2 hours, but occasionally swirl and invert the tube to mix.
4. Extract once with an equal volume of phenol
+
chloroform
+
isoamyl alcohol.
(The phenol, chloroform, and isoamyl alcohol should be in a 24:24:1 ratio. The isoamyl alcohol serves as
an antifoaming agent.)
5. Spin in a bench centrifuge for 5 minutes at room temperature.
6. Decant the aqueous layer with a Pasteur pipette into a new tube.
7. Extract twice more with phenol
+
chloroform
+
isoamyl alcohol. Respin and decant the aqueous layer
each time.
8. Extract the aqueous layer with chloroform and isoamyl alcohol (24:1). The interface between the organic
and aqueous layer should now be clean.
(Modified from Jowett 1986.)
Highly purified phenol is mixed with the sample under conditions that favor
the dissociation of proteins from the nucleic acids and the sample is then centri-
fuged (
Table 5.1
). Centrifugation yields two phases: 1) a lower organic phenol
phase carrying the protein, and 2) the less-dense aqueous phase containing the
nucleic acids. Some phenol-extraction protocols include chloroform; chloroform
denatures proteins, removes lipids, and improves the efficiency of the extrac-
tions. To reduce foaming caused by chloroform, isoamyl alcohol is usually added.
Phenol must be handled with great care and used only in a fume hood because
it is toxic. Extraction of DNA from cells or organisms should be carried out as
quickly as possible in ice with refrigerated buffers to minimize the activity of
nucleases in the cells that can degrade the DNA.
Kits that are purchased usually involve the following steps: 1) breaking the
cells open (cell lysis) to expose the DNA, by grinding or sonication; 2) remov-
ing membrane lipids by adding a detergent; 3) removing proteins by adding a
protease; 4) removing RNA by adding an RNase (not always done); and 5) pre-
cipitating the DNA with an alcohol, usually ice-cold ethanol or isopropanol,
resulting in a pellet upon centrifugation. In addition, a chelating agent may be
added to inactivate Mg
2
+
or Ca
2
+
to prevent DNases from degrading the DNA.
Some kits include chaotropic salts to disrupt cells.
5.3.2 DNA Extraction That Does Not Require Destroying the Specimens
It is often the case that retaining voucher specimens is important in ecological
and taxonomic studies. However, once the specimen(s) have been ground up
