Denaturing Polyacrylamide Gel Electrophoresis

Lisa M. Albright1, Barton E. Slatko2

1 null, Allison Park, Pennsylvania, 2 New England Biolabs, Beverly, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Appendix 3B
DOI:  10.1002/0471142700.nca03bs00
Online Posting Date:  May, 2001
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Abstract

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. Such gels are uniquely suited for nucleic acid sequence analysis, which is required, for instance, for all footprinting protocols. Thicker gels are often used to purify oligonucleotides. This appendix 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.

     
 
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Table of Contents

  • Basic Protocol 1: Pouring, Running, and Processing Denaturing Polyacrylamide Gels
  • Reagents and Solutions
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Pouring, Running, and Processing Denaturing Polyacrylamide Gels

  Materials
  • 70% ethanol or isopropanol in squirt bottle
  • 5% (v/v) dimethyldichlorosilane (Sigma) in CHCl 3
  • Denaturing acrylamide gel solution (see recipe)
  • TEMED
  • 10% (w/v) ammonium persulfate (make fresh weekly and store at 4°C)
  • 1× TBE electrophoresis buffer, pH 8.3 to 8.9 ( appendix 2A)
  • Samples for electrophoresis containing formamide and marker dyes
  • 30 × 40–cm front and back gel plates
  • 0.2‐ to 0.4‐mm uniform‐thickness spacers
  • Large book‐binder clamps
  • 60‐mL syringe
  • 0.2‐ to 0.4‐mm shark's‐tooth or preformed‐well combs
  • Sequencing gel electrophoresis apparatus
  • Pasteur pipet or Beral thin stem (Beral Enterprises)
  • Power supply with leads
  • 95°C heating block or water bath
  • 46 × 57–cm gel blotting paper (e.g., Whatman 3MM)
  • Kodak XAR‐5 X‐ray film
NOTE: Many companies provide equipment needed for sequencing experiments; a list of suppliers is provided in CPMB 3.0.1.
Table 0.b.1   MaterialsMigration of Oligodeoxy‐nucleo‐tides (Bases) in “Sequencing” Denaturing Polyacrylamide Gels Relative to Dye Markers

Polyacrylamide Bromphenol blue Xylene cyanol
5% 35 b 130 b
6% 26 b 106 b
8% 19 b 75 b
10% 12 b 55 b

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Figures

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Literature Cited

Literature Cited
   Maniatis, T., Jeffrey, A., and deSande, H.U. 1985. Chain length determination of small double‐ and single‐stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14:3787‐3794.
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