Acrylamide seems to be an ideal medium for isolating fragments less than 500 bp in length because the DNA tends of be more "biologically" active than DNA isolated from agarose gels. In our method, we run the DNA on a very thin acrylamide gel (0.2 - 0.3 mm thickness) containing TBE running buffer.  The region of gel containing the DNA is excised and transferred to a tube.  The DNA is allowed to diffuse out of the gel into a solution of TE plus 0.1M NaCl.  Salt is added or the solution is concentrated two fold.  Finally, the DNA is precipitated with ethanol.  Details for two situations are described below.

Isolating a nonradioactive PCR fragment.

1. Prepare several micrograms of fragment by PCR.  This amount can be detected by "UV shadowing".  Smaller amounts will require that you stain the sample with ethidium bromide.
2. Ethanol precipitate the PCR fragment and dissolve it in a total of 40 ul.  Add 20 ul of 6X Ficol dye.  It is important to use ficol to increase the density of the solution to ensure that the DNA runs as a sharp band on the acrylamide gel.
3. Prepare a 6% acrylamide gel containing 1X TBE.  The gel should be very thin (0.2 - 0.3 mm thickness), and should have wells that are approximately 2 cm wide by 1 cm deep.  After removing the comb from the gel, be certain to thoroughly clean the wells.  Dislodge any pieces of acrylamide with a piece of X-ray film and thoroughly rinse the wells with 1X TBE.  Assemble the gel in the electrophoresis apparatus.
 

	6%	8%	10%
29.2% Acrylamide/0.8% Bis	3 ml	4 ml	5 ml
10X TBE	1.5 ml	1.5 ml	1.5 ml
Water	10.5 ml	9.5 ml	8.5 ml
10% Ammonium persulfate	105 ul	105 ul	105 ul
Gently swirl solution before adding TEMED
TEMED	11.25 ul	11.25 ul	11.25 ul
Gently but thoroughly mix before puring the gel.  Allow the gel to polymerize for about 1 hour.

4. Just before loading the DNA, rinse the wells with 1X TBE by forcing buffer into the wells with a syringe.
5. Carefully load the DNA into each well.
6. Run the gel at 200 volts for 1 to 2 hours.
7. Separate the plates and cover the gel with Saran wrap.  Alternatively, you can leave the gel between the plates for a day without seeing much diffusion of the DNA.
8. Try detecting the DNA by shining the hand-held, short-wave length UV lamp on the gel.  Often, there is a small amount of fluorescence from the glass and the DNA can be detected as a shadow.  Outline the DNA with a marker.
• If no shadow is detected, peel the gel off the glass while leaving the gel attached to the saran wrap.  Sandwich the gel between pieces of saran wrap and place the gel on an intensifying screen (used with X-ray film) or a TLC plate.  These surfaces fluoresce more strongly than the glass plate and provide greater sensitivity for detecting a DNA shadow when illuminated with the UV light.
• If DNA shadows are still not observed, remove one layer of saran wrap and apply a thin layer of 1ug/ml ethidium bromide solution to the region of the gel where you expect to find your DNA.  Leave the gel uncovered for a few minutes and then overlay with saran wrap.  Detect the DNA with the hand-held UV lamp.
9. Excise the region of the gel containing DNA with a razor blade or scalpel.  Apply a downward motion to the blade to avoid ripping the gel into small shards.  Using fine forceps, remove the saran wrap and transfer the gel slice to a 1.5 microfuge tube.  Be certain not to include saran wrap since DNA sticks irreversibly to it.
10. Add 400 to 600 ul of TE + 0.1 M NaCl.  Place the tube at 37^oC for 6 to 18 hours to allow the DNA to diffuse out of the gel slice.  Alternatively, the gel slice can be stored in the refrigerator until there is time to elute the DNA.
11. To recover the DNA, microfuge the sample briefly to collect the gel at the bottom of the tube.  Transfer as much supernatant as possible to a fresh 1.5 ml tube.  Avoid transferring any of the gel.
12. At this point, you can concentrate the DNA and NaCl in the sample by doing several isobutanol extractions. This is recommended if the DNA has been stained with ethidium bromide as the extractions will remove the ethidium bromide.  For each extraction, add no more than 2 times the volume of sample currently in the tube in order to avoid extracting all the water.  Shake the tube for 2 minutes at room temperature and microfuge the tube at room temperature for 2 minutes.  Carefully remove and discard the upper phase, disposing of it in a bottle for isobutanol waste.  Continue doing extractions until the volume of the aqueous phase has been reduced to approximately 200 ul.
13. Extract 1 time with approximately 200 ul of ether.  The ether is stored in the cold cabinet, and you will find pipets in a small test tube taped to the left-hand wall of the hood for transferring the ether.  Add the ether to the sample and shake the tube for 30 to 60 seconds.  Microfuge for 2 minutes.  Carefully remove and discard the top layer consisting of ether.  Place the samples at 37^oC for 5 minutes to evaporate residual ether.
14. Adjust the final concentration of NaCl to 0.2 M and add 2 volumes of Ethanol.  Ice for at least 15 minutes, centrifuge in the cold at top speed for at least 20 minutes, and finally discard the supernatant.
15. Add 200 ul of cold 75% ethanol to the precipitate, briefly splash the solution around, microfuge in the cold for 5 minutes and discard the supernatant.
16. Air dry the DNA and dissolve it in 20 to 50 ul of TE.  Determine the concentration with the fluorometer.

1. Run the DNA on an acrylamide as described above.  It is often a good idea to ethanol precipitate the DNA at some point before loading it on the gel in order to reduce the amount of unincorporated counts that will be loaded on the gel.
2. After running the gel, remove the gel from the apparatus and set it aside.  Carefully place any radioactive buffer in the appropriate waste container and clean the apparatus.
3. Using a shield as much as possible, remove one plates from the gel and cover the radioactive gel with saran wrap.
4. Transport the following to the dark room: radioactive gel, waterproof marker, box of X-ray film, glass plate to press X-ray film against the gel.
5. Do the following under illumination from a safe light:
• Cut a piece of film to cover the region of gel containing DNA. Close the box containing the rest of the film (if you forget, you'll end up exposing the rest of the film in the box).
• Place the piece of film on the saran wrap covering the gel.  Hold the film in place and draw several lines across the edge of the film onto the saran wrap.  These line will serve to orient the film on the gel after it has been developed.
• Carefully place a piece of plexiglas on top of the film to hold the film flat against the gel.  Be certain not to disturb the alignment of the film with the lines drawn in the previous step.
• Expose the gel to film for 3 minutes - this is typical for radiolabelled DNA that will be used for footprinting or crosslinking.
• Develop the film.
6. With a razor blade, cut out the exposed bands on the film and use this as a template to guide the excision of the radioactive DNA.  It is critical to align the marks on the film with the marks on the saran wrap.  Also, there is usually some residual radioactive signal at the top of each lane on the film that should align with the well.
7. Excise the gel containing the radioactive DNA and isolate the DNA from the gel as described above.