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Method: Electroelution of DNA Fragments from Agarose into Dialysis Tubing

April 10, 1990

Matthew S. Holt


Purpose:

Time required:

  1. Restriction digest - minimum of 2 hours
  2. Electrophoresis - approx. 4 hours
  3. Elution - 2-5 hours depending on fragment size
  4. DNA Purification - 2 hours

Special Equiptment:

Procedure:

The restriction digest:

Electrophoresis:

  1. Generally, a 0.8 % TA gel will give sufficient separation for most digested fragments. Increase agarose to 1.2 % to isolate small fragments of 100 to 500 base pairs, decrease agarose to 0.6 % for fragments larger than 8 kb. 10 g of digested DNA will need to be spread out over approximately. 6 cm of wells. Some combs have 6 cm "slots" or wells or this can be achieved by taping up 8-10 individual wells. Individual wells can be used but some DNA will be lost due to the trailing up effect from the well edges.
  2. Run the gel at 60-80 volts until desired separation has occurred (depending on fragment sizes). Stain the gel for 30 minutes with ethidium bromide, but do not photograph gel yet because short wave UV can damage the DNA.
  3. Place Saran Wrap on the FOTODYNE Model 3-3500 UV light box. Transfer the gel to the Saran Wrap and turn on "PREPARATIVE" UV source (long wave). The fragments should become visible.
  4. Use a clean razor blade to cut above and below each fragment of interest minimizing the amount of agarose in the slice. Then free the slice by cutting the ends. Leave a small amount of the fragment ends in the gel to verify fragment sizes in the photograph. Minimize the amount of time the DNA is exposed to UV by having sterile labeled tubes ready for the slices.
  5. Photograph the gel after the fragment slices have been removed. Sometimes the fragment bands are too faint to be seen with long-wave and you must use short-wave. Again, try to minimize any UV exposure.

Elution:

  1. Cut a piece of dialysis tubing approximately 3 cm longer than the gel slice and clip one end. Gently push gel slice into open end and down to the clip. Add 300-500 ml of buffer (same as the gel, e.g., if the gel is 1 X TA, then use 1 X TA Buffer) so that the gel slice is completely immersed and there are no bubbles. Clip the open end (Note: gel slices will sit better in the electrophoresis box if clips are positioned identically).
  2. Place gel slices parallel to the electrodes and fill the electrophoresis box with buffer (again, same as original gel) until all tubing is submerged. Then remove some buffer until clip edges stabilize and rest on bottom. Electroelute at 80-100 volts for 2-5 hours, longer for large fragments. Monitor the movement of the DNA with a hand held UV source (long-wavelength).
  3. DNA may stick to dialysis tubing after current flow. Either reverse the electrodes and run for 15 seconds to dislodge the DNA or be careful to resuspend the DNA before removing the buffer. To resuspend DNA inside the tubing, first open one clip and carefully remove the agarose slice (which can be restained to verify elution) without losing any buffer. Then replace the clip and with your fingers, press along the length of tubing to mix the DNA with the buffer. Open the clip and remove the buffer with a pipetman. If low yields are suspected, add another 100 l 1 X buffer to the empty tubing and repeat these steps to rinse out the tubing. Final volume should be less than 600 l. If the agarose slice is much longer than 4 cm, the amount of buffer retrieved may be much more than 600 l. In this case split the DNA buffer into two or three tubes so that no tube has more than 600 l.

DNA cleaning and precipitation:

  1. Add 1 volume (500 l) of phenol and mix (NOTE: hard vortexing will shear large fragments). Spin in microcentrifuge at 14,000 rpm (at 4 degrees C or at room temperature) for five minutes. Phenol sinks and the DNA (in the aqueous phase) will be on top. Remove the top aqueous phase without pulling any debris at the interphase (don't try to get it all - the DNA quality will be better). Place the aqueous phase in clean eppendorf tubes.
  2. Add 1 volume (500 l) of chloroform, mix, and spin in microcentrifuge and 14,000 rpm for two minutes. Chloroform will sink and the DNA will be in the aqueous phase. Remove the top aqueous phase leaving behind any debris at the interphase and transfer to clean eppendorf tubes.
  3. Add 1/10 volume (50 l) 3M Na Acetate and 800 l 95% EtOH ( EtOH must be at -20 degrees C, or place tubes in the -20 degrees C freezer for 15 minutes after adding EtOH). Spin in microcentrifuge at 14,000 rpm, 4 degrees C for 30 minutes. Decant supernatant and add 500 l 70% EtOH (must be cold!) to the pellet. Spin again for five minutes, decant the ethanol wash and invert the tubes to dry. For low yields or barely visible pellets, speed vacuum drying is recommended.
  4. When the DNA pellet is dry, add 20-50 l 1 X TE ; the pellets should resuspend fairly easily. Quantitate on a mini-gel with several lambda DNA standards and 1kb ladder for sizing.

Safety Precautions:

References:

Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp. 6.29.