Bio-Rad Model 422 Electro-Eluter - Manual (Page 12)

Section 7
Visualization of DNA
7.1 Visualizing DNA in Agarose Gels
1. Electrophorese the DNA in an agarose gel. Use high quality agarose to prevent agarose
contaminants from affecting subsequent manipulations.
2. Stain the gel with an ethidium bromide solution. The concentration of the stain should be
0.5 µg/ml.
Caution: Ethidium bromide is a mutagen. Use gloves when handling ethidium bromide.
3. Place the stained gel on a UV light box. Cut out the band of interest with a scalpel.
7.2 Visualizing DNA in Acrylamide Gels
Often small pieces of DNA need to be eluted. For DNA of less than 1,000 base pairs,
acrylamide gels are recommended.
1. Electrophorese the DNA in a polyacrylamide gel.
2. After electrophoresis, stain the DNA using ethidium bromide at 0.5 µg/ml in 1x TBE.
Wear gloves when using ethidium bromide; it is a mutagen.
Note: Polyacrylamide quenches the fluorescence of ethidium bromide. Therefore, at least
10 ng of DNA/band must be loaded.
3. Excise the band of interest. Follow the Model 422 Electro-Eluter assembly procedures
given in Section 4.
Section 8
DNA Elution
8.1 Elution from Gel
1. Soak the membrane caps for at least 1 hour at 60 °C in DNA elution buffer. See Section
9 for recipes. Membrane caps may be soaked for longer than 1 hour. Wear gloves when
handling the membrane caps to prevent the dialysis membrane from becoming contam-
inated. Use Green Membrane Caps MWCO 3500 daltons (catalog number 165-2986).
2. Assemble the Model 422 Electro-Eluter as described in Section 4.
3. The elution is done at ~10 mA/Glass Tube constant current. Elute the DNA for 15 min-
utes to 1 hour. The optimal elution time is dependent on the size of the DNA fragment
being eluted. Elution times greater than the optimal time tend to drive the DNA into the
dialysis membrane, making it difficult or impossible to retrieve the DNA.
4. After elution, reverse the polarity for approximately 1 minute to remove the DNA from
the dialysis membrane. Take the electro-eluter module out of the buffer tank. Take the
module, with the Glass Tubes attached, to a sink. If a stopper has been used, remove the
stopper from the upper buffer chamber and allow the buffer to drain out. If there are no
stoppers, carefully pull one Glass Tube out of the upper buffer chamber and allow the
upper buffer to drain.
5. Using a plastic pipet, remove the buffer left in the tube down to the level of the frit and
discard the liquid. Work quickly and carefully. Do not dislodge the Silicone Adaptor or
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