A29 Electroelution

To recover DNA or fragments of DNA from a gel.

Prior Work

A gel must be available containing DNA (or protein). The DNA would have been separated during a restriction analysis (A-28).

Materials

• A Low Conductivity Buffer, 0.005x TEA
  • 1x TEA
  • 50 mM Tris OH
  • 0.1 mM EDTA
    • Adjust pH to 7.8 with acetic acid

Background

• Electroelution involves removing DNA (or sometimes protein) from a piece of gel obtained from a gel slab on which a DNA separation has been previously performed. This might follow a restriction analysis, for example. Under normal circumstances, the DNA will have been stained with ethidium bromide. When examined with 302 nm UV, the ethidium bromide fluoresces visibly. A longer wavelength radiation, say 360 nm, reduces nicking due to the UV radiation and increases yield. DNA electrophoresed in the presence of ethidium bromide might have been broken so if that DNA is used it might not be in its active conformation.
• Either of two devices may be used to achieve elution of the DNA. Both involve passing an electric current through the piece of gel. The gel is immersed in a low conductivity buffer. The charged DNA molecules are favored as charge carriers. As these polyelectrolyte ions carry charge, they migrate and eventually emerge from the gel matrix.
• In one device, the DNA ions are stopped by a membrane barrier which permits small but not large ions to penetrate its pores. In the second device, the current is routed through a small, "Vee"-shaped opening into which very concentrated salt solution is placed. When the DNA ions reach this salt solution, many ions become available to carry charge. Also, the smaller buffer ions are more mobile. Thus the DNA ions enter the buffer at one side, but remain in this region because they are no longer major contributors to the electrical conduction through the system.
• Electroelution can be used to obtain proteins from gels. The proteins should be only lightly stained and not fixed if elution is to be successful. Also, attention needs to be paid to the pH of the buffers which should be about 1.5-2 units away from the protein pI. The protocols for electrode polarities will depend upon the proteins as well.

Procedure

1. Wear a face shield for protection from UV radiation. UV light makes the bands visible. Use a razor blade to cut out the desired bands. Remove the pieces of interest with forceps or a spatula. Then transfer the pieces to plastic wrap. Speed during this step is essential -- reducing the exposure to UV reduces nicking and increases yield.
2. The first device demonstrated in the movie uses a membrane partition. Read the manufacturer's instructions. Fill the end tanks with high conductivity buffer. Fill the middle tanks with lower conductivity buffer. The sample cups are soaked. Select a soaked cup. Place the sample gel in the large well of the cup. The bottom of each well consists of dialysis membranes. (Some workers have used this cup device to accomplish small scale dialyses. With great care, the plastic ring at the bottom of the small well may be removed, and fresh membranes installed.) Fill the device with low conductivity buffer. Wear gloves -- the gel contains ethidium bromide. The transfer of the gel from the forceps to the well often is facilitated by adding low conductivity buffer first. Place the cup device into the tank.
3. Install the cover, connect the cords, and turn on the power. Apply current for the recommended period of time. Turn off power, and disconnect power cords. (Some workers perform one additional step. When the elution is complete, and the power is turned off, the leads are reversed and power applied for 60 seconds. This has the effect of removing any DNA adsorbed on the membrane at the bottom of the well.)
4. Remove the tank top and the cup. The DNA should have migrated to the bottom of the small well. Use a Pasteur pipet to remove buffer from the bridge connecting the wells. Use a Pasteur pipet to remove the buffer from the top of the small well.
5. The small extension at the bottom of the small cup contains the DNA. Use a plastic pipet or micropipettor to remove the DNA solution. (This prevents membrane puncture.) Use a forceps to remove the gel. Check the gel under UV light to be certain that the fluorescence has been removed.
6. An alternative electroelution procedure follows. Read the manufacturer's instructions. Place buffer (0.5x TBE) in the tanks on each side of the "Vee" tube. Add 7.5 M ammonium acetate solution containing a dye marker such as bromphenol blue to the "Vee" tube.
7. Wear gloves. Place a piece of gel containing desired DNA in the cup opposite the "Vee" tube. Replace the cover.
8. Connect the power cords to the tank and the power supply. Apply current for the recommended period of time. Turn power off. Disconnect the power cords from the power supply and tank.
9. Remove the tank cover. Press the valve. Drain the tanks.
10. (Check that the outlet tube drains to a sink or collection vessel.) Use a forceps to remove the gel. Check the gel under UV light to be certain that the fluorescence has been removed. Use a pipet to withdraw the liquid from the "Vee" tube and transfer it to a microcentrifuge tube. The eluted DNA will be in the microcentrifuge tube.

Going Further

The DNA might be used in ligation (A-21).

References