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KLAUS DEICHMANN
Subprotocol 3
Denaturing Polyacrylamide Sequencing Gels
The following protocol describes casting and running a standard polyacrylamide sequencing gel. Quality and running conditions are as important for
the outcome of sequencing as the sequencing reaction itself. No protocol
will be appropriate for all purposes, so the length of the gel as weil as the
polyacrylamide concentrations will differ with the particular application.
Materials
Reagents
• Acrylamide
• N ,N'- methylenebisacrylamide
• Urea (ultrapure)
• TEMED
• 10% Ammonium Persulfate
• Dichlorodimethylsilane (e.g. Methyl-SilaneTM, LKB)
• Amberlite MB1
Solutions
• Ultrapure H 2 0
• 10% acetic acid/10% methanol in water
• 10% methanol in water
• 10 x TBE buffer (121 g Tris base, 7.4 gEDTA, 53.4 gBoric acid in 1000 ml
A.D., pH adjusted to 8.3)
Supplies
• Sequencing gel apparatus
• vacuum gel-drying apparatus
• glass plates (34 x40 and 34 x38 cm for conventional sequencing gel)
• 2 spacers, 0.4 mm thick
• shark's tooth combs
• tray (40 x50 cm)
• filter paper (e.g. Whatman No.l)