DNA Denatured PAGE/UREA PAGE
@DrMuhammadImranOfficial
Thin polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (500 nucleotides) single-stranded fragments of DNA or RNA that differ in length by as little as one nucleotide
Thicker gels are often used to purify oligonucleotides.
The following protocol describes the pouring, running, and processing of a typical “sequencing” gel which is 40-cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide. RNA samples are sometimes run on gels prepared with 8 M urea.
Applications
1. RT PCR
2. Simple Sequence Length Polymorphism
3. Mutational Analysis
4. Genetic syndrome
5. Forensic Science
1. Meticulously wash front and back 30 × 40–cm gel plates with soap and water. Rinse well with deionized water and dry. Wet plates with 70% ethanol or isopropanol in a spraying bottle and wipe dry with Kimwipe or other lint-free paper towel. A typical preparative gel uses 20 × 16–cm plates.
2. Apply a film of 5% dimethyldichlorosilane in CHCl3 to one side of each plate by wetting a Kimwipe with the solution and wiping carefully. After the film dries, wipe plate with 70% ethanol or isopropanol and dry with a Kimwipe. Check plates for dust and other particulates
3. Assemble gel plates according to manufacturer’s instructions, with the silanized surfaces facing inward. Use 0.2- to 0.4-mm uniform-thickness spacers and large book-binder clamps, making certain side and bottom spacers fit tightly together.
Use thicker spacers (e.g., 1.6-mm) for preparative gels
4. Prepare 60 mL of desired denaturing acrylamide gel solution in a 100-mL beaker.
To speed dissolution of urea, the gel mix can be heated before adding TEMED and ammonium persulfate; however, to prevent degradation of acrylamide, do not heat over 55°C. Allow to cool to room temperature (25°C) before adding the TEMED and ammonium persulfate to prevent polymerization while pouring the gel. If particulate matter remains, filter through a Whatman no. 1 filter paper in a funnel. To achieve slower polymerization, reduce amounts of TEMED and ammonium persulfate to 40 µL and 0.4mL, respectively
5. Pour gel immediately. Gently pull acrylamide solution into a 60-mL syringe, avoiding bubbles. With short plate on top, raise upper edge of gel sandwich to 45° angle from the benchtop and slowly expel acrylamide between plates along one side. Adjust angle of plates so gel solution flows slowly down one side.
6. When solution reaches top of short plate, lower gel sandwich so that the top edge is 5 cm above benchtop. Insert Comb. Use book-binder clamps to pinch combs between plates so that no solidified gel forms between combs and plates. Layer extra acrylamide gel solution onto comb to ensure full coverage.
7. When gel polymerizes, remove bottom spacer or tape at bottom of gel sandwich.
8. Fill bottom reservoir of gel apparatus with 1× TBE buffer so that gel plates will be submerged 2 to 3 cm in buffer. Place gel sandwich in electrophoresis apparatus and clamp plates to support.
Sweep out any air bubbles at bottom of gel by squirting buffer between plates using syringe with a bent 20-G needle.
9. Pour 1× TBE buffer into top reservoir to 3 cm above top of gel. Rinse top of gel with 1× TBE buffer using a Pasteur pipet.
10. Reinsert teeth of cleaned shark’s-tooth comb into gel sandwich with points just barely sticking into gel. Using a Pasteur pipet or Beral thin stem, rinse wells thoroughly with 1× TBE buffer to remove stray fragments of polyacrylamide. If a preformed-well comb is used, this step is omitted.
Remove extraneous polyacrylamide from around combs with razor blade. Clean spilled urea and acrylamide solution from outer plate surfaces with water. Remove shark’s-tooth comb gently from gel sandwich without stretching or tearing top of gel.
Clean comb with water so it will be ready to be reinserted in step 10. If preformed-well comb was used, take care to prevent tearing of polyacrylamide wells. This comb will not be reinserted.
12. Rinse wells with 1× TBE buffer just prior to loading gels, to remove urea that has leached into them.
14. Run gels at 45 to 70 W constant power. Maintain a gel temperature of 65°C. Observe migration of marker dyes (Table A.3B.2) to determine length of electrophoresis.
Temperatures 65°C can result in cracked plates or smeared bands; too low a temperature can lead to incomplete denaturation.
13. Heat samples 2 min at 95°C in covered microcentrifuge tubes, then place on ice. Load 2 to 3 µL sample per well. Rinse sequencing pipet tip twice in lower reservoir after dispensing from each reaction tube.