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Method: Electroelution of DNA Fragments from Agarose into Dialysis Tubing
April 10, 1990
Matthew S. Holt
To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.
- Restriction digest - minimum of 2 hours
- Electrophoresis - approx. 4 hours
- Elution - 2-5 hours depending on fragment size
- DNA Purification - 2 hours
- SPECTRA/POR dialysis tubing (VWR, B, F 10mm x 50 ft., cat.#132645) and clips
The restriction digest:
Use 5-10 µg DNA with the appropriate enzyme digest (see Restriction Digest Methods) for a minimum of 2 hours, maximum of overnight. If available, use a restriction enzyme map of the DNA to be digested to determine what enzyme gives the best fragment separation and what the size of the fragments of interest should be. For example, cutting a plasmid into two similar size fragments makes separating the fragments difficult. The restriction maps can identifyanother enzyme which only cuts one of these fragments (usually the vector). By cutting with both enzymes, the unwanted fragment can be cut into smaller pieces giving better isolation of the wanted fragment. NOTE: It is always helpful to run a small aliquot of the digest (250-500 ng) on a mini-gel to visualize the fragment pattern and determine how long to run the gel to get the desired separation, as well as to test for complete digestion.
- 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 µg 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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).
- 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 µl 1 X buffer to the empty tubing and repeat these steps to rinse out the tubing. Final volume should be less than 600 µl. If the agarose slice is much longer than 4 cm, the amount of buffer retrieved may be much more than 600 µl. In this case split the DNA buffer into two or three tubes so that no tube has more than 600 µl.
DNA cleaning and precipitation:
- Add 1 volume (500 µl) 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.
- Add 1 volume (500 µl) 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.
- Add 1/10 volume (50 µl) 3M Na Acetate and 800 µl 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 µl 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.
- When the DNA pellet is dry, add 20-50 µl 1 X TE ; the pellets should resuspend fairly easily. Quantitate on a mini-gel with several lambda DNA standards and 1kb ladder for sizing.
Ethidium bromide is a powerful mutagen and moderately toxic. Gloves should be worn when handling gel and gel slices. UV can cause severe burns. Always wear eye protection. Phenol can cause severe burns and gloves should be worn. Phenol waste must be contained and disposed of through WUMS Hazardous Waste Department. Chloroform is a carcinogen.
Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp. 6.29.