Denaturing polyacrylamide electrophoresis is a useful method to purify chemically synthesized oligonucleotides from 2 to 200 nt long, depending on the percentage of PAGE chosen. DNA/RNA samples are loaded onto a urea-based denaturing gel, separated by electrophoresis, and finally recovered from the crushed gel slice.
10x and 1x TBE buffer, pH 8
40% acrylamide stock solution (38% acrylamide/2% bisacrylamide, 19:1)
10% ammonium persulfate (APS, less than 2 months old, store at 4°C)
urea loading buffer
3M sodium acetate,pH 5.2 or 3M sodium chloride
Thin-layer chromatography (TLC) plate with fluorescent indicator (e.g., Silica Gel F-254 or IB-F)
Glass plates, spacers, and combs for gel preparation
Acrylamide gel electrophoresis apparatus
0.2 micron filter (Gelman Sciences)
Additional reagents and equipment for phenol extraction and ethanol precipitation.
Prepare the sample
1. Follow the appropriate deprotection protocol to prepare the DNA/RNA sample for electrophoresis.
Be sure to dry the sample through freeze drying or speed vac to get off-white powder sample. High concentration of salt (especially Mg2+) in DNA/RNA samples might cause problem during gel running. Ethanol precipitation is recommended if the sample contains too much salt as the yellowish liquidor crusty pellet. Generally the sample is resuspended in 0.5 ml distilled water and add 1/10 volume 3.0 M sodium acetate or sodium chloride. Add 3 volumes of absolute ethanol to precipitate by chilling to -80 °C for approximately 30 minutes. Centrifuge for 20 minutes at 16000x g and 4°C. Decant and save the supernatant. The pellet can be washed by 70% ethanol, and dried.
Prepare the gel
2. Assemble the gelcasting apparatus.
Gel spacer and casting systems are recommended, or the bottom spacer and clamps are necessary to block the acrylamide solution leaking. Clean the gel plates thoroughly by soapy water and ethanol. For analytical gel (thickness 0.8 or 0.4 mm), silanizing one or both of the plates will help the gel preparation and the post-electrophoretic separation of the gel from the plate.
3. Prepare the gel solution (see Table 1 for appropriate acrylamide concentrations for resolving single stranded DNAs). For a denaturing acrylamide gel of 20 cm x 20 cm x 1.5 mm, 60 ml of gel solution is sufficient, and it can be made by mixing the following:
25.2 g urea (final concentration of 7 M)
6 ml 10x TBE buffer
desired amount of 40% acrylamide stock solution
water to a final totalvolume of 60 ml
Pick a concentrationof acrylamide that will allow the single stranded nucleic acid to migrate approximately one-half to three-fourths the way through the gel when the loading dye has reached the bottom of the gel. This allows for good separation of non- and full-length products.
Use a flask that has a wide mouth and a spout for pouring.
Acrylamide (%) Fragment sizes separated (bases) migration of bromophenol blue (bases) migration of xylene cyanol (bases)
30 2 to 8 6 20
20 8 to 25 8 28
10 25 to 35 12 55
8 35 to 45 19 75
5 70 to 300 35 130
Urea loading buffer
8 M urea
20 mM EDTA
5 mM TRIS pH7.5
0.5 % dye by mass, either xylene cyanol, bromophenol blue, or both.
add one volume of loading buffer to sample if a solution or enough to dissolve a powder.
4. Heat the mixture to completely dissolve the urea.
5. Add 40 ml of TEMED and swirl the flask to insure thorough mixing. Immediately add 300 ml of 10 % APS and mix thoroughly. Polymerization has begun so all SUCCEEDING steps must be PERFORMED promptly. Pour the acrylamide between the gel plates and insert the comb. Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allowthe gel to polymerize for approximately 1 h.
Run the gel
6. After polymerizationis complete, remove the comb and any bottom spacers from the gel. Washthe gel plates free of spilled acrylamide and be sure that the spacers are properly seated and clean.
7. Position the gel according to the lower tank and top tank. Fill the reservoirs of the electrophoresis tanks with 1X TBE, so that the wells are covered. A syringe with a bent needle may be used to remove air bubbles trapped under the gel that will disrupt the current flow.
8. Use a DC power supply to pre-run and warm the gel for a least 30 minutes at 20-30 V/cm.
9. Resuspend the oligonucleotide pellet obtained from step 1 in 1X urea loading buffer by heating it at 90°C for 2 minutes. The amount of sample that can be loaded depends on the efficiency of the synthesis reaction.
10. Rinse the wells thoroughly with 1XTBE solution immediately prior to gel loading.
The 7 M urea dissolvedin the gel will start to diffuse from the wells thereby creating a dense layer at the bottom of the wells that prevents sample loading and decreases resolution. Rinsing eliminates this problem.
11. Load the samples.
Tracking dyes such as bromophenol blue and xylene cyanol may be added to the samples or in empty lanes to monitor migration.
12. Electrophorese the gel at 20-30 V/cm (constant voltage) until the tracking dyes indicatethat the oligonucleotide has migrated one-half to three-fourths the way through the gel.
The current in the circuit and the heat generated for higher percentage gels (>15 %acrylamide) are corresponding smaller since the increased acrylamide concentration leads to greater resistance. While some heating of the gel during electrophoresisis desirable since it helps to denature the sample, all gels should be monitored to make sure that they do not generate so much heat that the plates crack.
13. When the oligonucleotideis sufficiently resolved, turn off the power supply and detach the platesfrom the electrophoresis tank. Pry off the top plate. Cover the gel with plastic wrap (taking care to avoid bubbles and folds) and invert the plate onto a TLC plate with a fluorescent indicator. Using a spatula, peel acorner of the gel away from the plate and onto the plastic wrap. Pry off the remaining plate and place another sheet of plastic wrap on top of the gel.
Recover the oligonucleotide
14. Visualize the bands on the gel by briefly exposing them to short-wave ( 254 nm) radiation from a handheld lamp. The bands will appear as black shadows on a green background. Outline the bands using a marking pen.
The desired band is generally the darkest one on the gel (excluding material that runs at the dye front); it should also be the slowest migrating band unless deprotection was incomplete. Lighter bands containing partially protected oligonucleotides if they are present will migrate considerably above the major fully deprotected band. If the stepwise efficiency of the synthesis is low, a smear may be seen instead of a clear band. Cut out the top of the smear.
Avoid unnecessarily long UV exposure which will damage the nucleic acids.
15. Cut out the bands directly with a clean scalpel or razor blade.
16. Chop the gel slabs into fine particles in a tube.
17. Add appropriate amount of buffer or water to the gel slab. Freeze the sample for 30 minutes at -80°C or until frozen solid. Quickly thaw it in a hot water bath and let soak for 5 minutes at 90°C. Elute on a rotary shaker overnight at room temperature.
This freeze-rapid thaw approach greatly decreases elution time and increases yield by allowing ice crystals to break apart the acrylamide matrix. Since elution is a diffusion-controlled process, more water or buffer will aid in elution efficiency. Also, note that longer oligonucleotides will take longer to diffuse from the gel. If speed is essential and high yields are dispensable, enough sample can be obtained for most experiments in only a few hours of extraction. Increasing the temperature to 37°C will also speed the process. Yield may be increased upon repeated elutions.
18. Spin the tube to pellet the gel fragments and use a pipette to remove the supernatant. Remove the unnecessary salt by ethanol precipitation or Glen-pak desalting cartridge.
19. Redissolve the oligonucleotide in water or buffer if appropriate.