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— ~4 hours
- Elution—2-5 hours depending on fragment size
- DNA purification—2 hours.
- SPECTRA/POR dialysis tubing (VWR, B, F 10 mm × 50 ft., cat.#132645) and
The restriction digest. Use 5–10 µg DNA with the appropriate enzyme digest for
a minimum of 2 hours, maximum of overnight. If available, use a restriction
enzyme map of the DNA to be digested to determine which enzyme provides
the best fragment separation and what the size of the fragments of interest
should be. For example, cutting a plasmid into 2 similar-size fragments makes
separating the fragments difficult. The restriction maps can identify another
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.
It is always helpful to run a small aliquot of the digest (250–500 µg) on a minigel
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 provide 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. spread
out 10 µg of digested DNA 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 longwave and you must
use shortwave. 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.
Gel slices will sit better in the electrophoresis box if clips are positioned
- 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 handheld UV source (longwavelength).
- 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 1X 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 2 or 3 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 14000 rpm (at 4°C or at room
temperature) for 5 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 14000 rpm for 2 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°C, or place tubes in the –20°C freezer for 15 minutes after
adding EtOH). Spin in microcentrifuge at 14000 rpm, 4°C for 30 minutes.
Decant supernatant and add 500 µL 70% EtOH (must be cold!) to the
pellet. Spin again for 5 minutes, decant the ethanol wash, and invert the
tubes to dry. For low yields or barely visible pellets, speed vacuum drying
- When the DNA pellet is dry, add 20–50 µL 1X TE; the pellets should
resuspend fairly easily. Quantitate on a mini-gel with several lambda DNA
standards and a 1-kb 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 the Hazardous Waste
Department. Chloroform is a carcinogen.