Bio-Rad Model 422 Electro-Eluter - Manual (Page 13)

the membrane cap. Make sure that the liquid below the frit is not disturbed or shaken up
during this process.
6. Remove the Silicone Adaptor together with the membrane cap from the bottom of the
Glass Tube. The buffer level should be at the top of the membrane cap. Using a clean
plastic pipet, pipet the liquid remaining in the membrane cap into a microfuge tube. The
volume should be approximately 400 µl. Rinse the membrane cap with 200 µl of fresh elu-
tion buffer. Add this liquid to the centrifuge tube which contains the eluted DNA. The vol-
ume collected will be approximately 600 µl. Do this for each Glass Tube.
7. Typical elution yields for DNA are between 70% and 90%. Some DNA will be irre-
versibly bound to the dialysis membrane, even if the polarity is reversed at the end of
electro-elution.
8. Membrane caps should not be reused for DNA unless the same fragment is being eluted.
Contamination of different DNAs will occur if a membrane cap is used for more than
one fragment, because the DNA from the first elution will stick to the membrane.
9. If the same fragment of DNA will be eluted later, the membrane caps can be reused. Store
the caps in DNA elution buffer with 0.05% sodium azide (NaN
3
) in the refrigerator. It is
not necessary to reheat or resoak them after the first use.
8.2 DNA Purification
After the DNA has been eluted and collected, it will have to be purified for further use.
1. Add 1/10 volume 3M sodium acetate (NaOAc) to the DNA sample. Add DNase free
tRNA to the eluted DNA. The concentration of nucleic acids should be approximately
10 µg/ml. The tRNA is added as a carrier to insure that all of the DNA will be ethanol pre-
cipitated.
2. Extract the DNA with an equal volume of equilibrated phenol. Vortex for at least 30 sec-
onds. Spin in a microfuge for 2 minutes. Pull off the aqueous phase and put it in a new
microfuge tube.
3. Extract a second time with an equal volume of phenol/chloroform. The chloroform is a
24:1 mixture of chloroform and isoamyl alcohol. Vortex for at least 30 seconds. Spin in
a microfuge for 2 minutes. Pull off the aqueous phase and put it in a new microfuge tube.
4. Add 2 to 2.5 volumes of ice-cold ethanol. Place at -70 °C for one half hour, or overnight
at 20°C.
5. Spin down for 15 minutes in a cold microfuge. Decant the supernatant.
6. Resuspend in 100 µl of 10 mM Tris pH 7.6, 1 mM EDTA (TE). Repeat the ethanol pre-
cipitation. Decant the supernatant and rinse the pellet with 70% ethanol. Allow the sam-
ple to dry. Resuspend in TE.
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