6 Denaturing Gradient Gel Electrophoresis
89
for experimentally elucidating melting domains within a given DNA fragment. Only one sample can be separated per gel. Parallel DGGE can separate
many samples on one gel and is therefore the method of choice for mutation
screening.
Materials
• Gradient maker (15-25 ml capacity per side), with silicone tube (4 mm Supplies
external diameter)
• Magnetic stirrer
• Gel-casting stand
• Vertical electrophoresis unit with heat exchanger (51 buffer volume) to
hold 16 x 18 cm gels, 0.75 mm thickness (e.g. Hoefer Scientific SE600)
• Heating water bath with external circulation, silicone tubes (12 mm external diameter)
• Power supply
• Peristaltic pump
• Variable clamp for flow adjustment in silicone tubes; 20 gauge needle
• Polyacrylamide and bis-acrylamide 38.4:1.6 solution (e.g. Roth)
• Urea p.a. grade
• Formamide, deionized, p.a. grade; in our experience no deionization is
necessary for up to 6 months if stored in the original vessel. If deionization is required, mix Dowex XG8 mixed-bed resin with formamide 1:20
w/v, stir gently for 30 minutes, carefully remove the formamide avoiding
the resin beads and store from light protected at -70°C.
• 50x TAE buffer (2M Tris, 50 mM EDTA, adjust pH with acetic acid to 8.0)
• 10o/o ammonium persulfatein water, prepared fresh daily
• TEMED
• Ultrapure water
• 6x gelloading buffer (0.25o/o bromophenol blue, 30o/o glycerel in water)
• DNA fragment in water or TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM
EDTA), or PCR reaction buffer. Best results are obtained with fragments
Reagents
and solutions
89
for experimentally elucidating melting domains within a given DNA fragment. Only one sample can be separated per gel. Parallel DGGE can separate
many samples on one gel and is therefore the method of choice for mutation
screening.
Materials
• Gradient maker (15-25 ml capacity per side), with silicone tube (4 mm Supplies
external diameter)
• Magnetic stirrer
• Gel-casting stand
• Vertical electrophoresis unit with heat exchanger (51 buffer volume) to
hold 16 x 18 cm gels, 0.75 mm thickness (e.g. Hoefer Scientific SE600)
• Heating water bath with external circulation, silicone tubes (12 mm external diameter)
• Power supply
• Peristaltic pump
• Variable clamp for flow adjustment in silicone tubes; 20 gauge needle
• Polyacrylamide and bis-acrylamide 38.4:1.6 solution (e.g. Roth)
• Urea p.a. grade
• Formamide, deionized, p.a. grade; in our experience no deionization is
necessary for up to 6 months if stored in the original vessel. If deionization is required, mix Dowex XG8 mixed-bed resin with formamide 1:20
w/v, stir gently for 30 minutes, carefully remove the formamide avoiding
the resin beads and store from light protected at -70°C.
• 50x TAE buffer (2M Tris, 50 mM EDTA, adjust pH with acetic acid to 8.0)
• 10o/o ammonium persulfatein water, prepared fresh daily
• TEMED
• Ultrapure water
• 6x gelloading buffer (0.25o/o bromophenol blue, 30o/o glycerel in water)
• DNA fragment in water or TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM
EDTA), or PCR reaction buffer. Best results are obtained with fragments
Reagents
and solutions