Fall 2015
Plant DNA extraction
1.
Place the sample in a sterile mortar. Use a pestle with liquid nitrogen to grind the tissue,
leaving no large tissue fragments.
2.
Transfer approximately 250 μl (or less) of the sample to a labeled “eppendorf” (1.5 mL)
tube, and add 500 μl of CTAB buffer with proteinase K. Mix well.
3.
Place the (LABELED) tube in a 60oC heat block or water bath for 30 minutes (mix every
10 minutes). Label the tube with an appropriate name that you'll be able to recall as your own.
4.
Add 500 μl 24:1 chloroform:isoamyl alcohol to each tube. This should be performed in
the hood. The chloroform:isoamyl alcohol separates the sample solutions into different phases,
partitioning DNA into the upper (aqueous) layer, while organics are separated into the lower
phase. Proteins are often found as a band in between the two layers.
5.
Centrifuge the tubes using max RPM for 10 minutes. While the samples are spinning,
prepare new, sterile eppendorf tubes labeled the same as your originals.
6.
After spinning, be careful not to disturb the layers and pipette the top (aqueous) layer into
a new tube prepared in the last step. Avoid the protein that is at the interface.
7.
Dispose of the remaining 24:1 chloroform:isoamyl alcohol in the fume hood. Liquid
waste can be poured into the waste bottle, while the tubes should be disposed of in the jar.
8.
Add an equal (or slightly greater) volume of isopropanol to the tube (~ 400 μl). The
isopropanol causes the DNA to precipitate.
9.
Centrifuge your samples using max RPM for 5 min.
10.
Immediately, decant the isopropanol into a waste container. The DNA should now be in a
pellet (which may or may not be visible) near the bottom of the back of the eppendorf tube (tab
side - this is why it's important to orient your centrifuge tubes with the tabs to the outside of the
rotor). Add 750 μl of 76% EtOH/NH4O-Ac solution and mix by inverting. Centrifuge at max
RPM for 5 min.
11.
Remove the 76% EtOH/NH4OAc by decanting and add 500 μl of 75% EtOH. Mix by
inverting and centrifuge at max RPM for 5 min. The 75% EtOH cleans the DNA of excess salts
that are present.
15.
Remove the EtOH by decanting and use a 200 μl pipette tip to remove any remaining
liquid (carefully - away from the back of the tube where the DNA should be pelleted). Leave the
cap open to allow the DNA pellet to dry (the more liquid you remove in the previous step, the
quicker this will happen) and place the tube in the 37oC incubator until there is clearly no more
liquid present (~3-8 minutes). Do not over dry the pellet by leaving it in the incubator after the
liquid has disappeared.
16.
When completely dry, dissolve the pellet in 50 μl of sterile water. Pipetting the water up
and down quickly and repeatedly usually accomplishes this, along with some gentle flicking of
the tube bottom.
Solutions
1. CTAB Buffer: 2% CTAB, 1.4 M NaCl, 100 mM Tris pH 8, 20 mM EDTA, 0.2% (v/v) beta
mercaptoethanol, adjusted to pH 8, proteinase K
2. chloroform
3. Ethanol/NaOAc solution (76% (v/v) EtOH, 10 mM NaOAc
4. absolute ethanol
5. 75% ethanol