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 Human Genetics
Unit Number:  Appendix 3F
DOI:  10.1002/0471142905.hga03fs00
Online Posting Date:  May, 2001
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Abstract

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 commonly used for DNA sequence analysis, as well as in PCR amplification of SSLPs (simple sequence length polymorphisms) for genotyping, genotyping by the ligase chain reaction (LCR), analysis of mutations by RNase A cleavage, chemical cleavage of heteroduplex DNA to identify mutations, and molecular analysis of fragile X syndrome by PCR. This appendix presents a protocol for the pouring, running, and processing of a typical 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
  • Tables
     
 
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Materials

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

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • 70% ethanol or isopropanol in squirt bottle
  • 5% (v/v) dimethyldichlorosilane (Sigma) in CHCl 3
  • recipeDenaturing acrylamide gel solution (see recipe)
  • TEMED
  • 10% (w/v) ammonium persulfate (make fresh weekly and store at 4°C)
  • 1× TBE buffer, pH 8.3 to 8.9 ( appendix 2)
  • Samples for electrophoresis containing formamide and marker dyes (e.g., units 2.5, 2.6, 7.2, 7.6, or 9.5)
  • 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
CAUTION: Dimethydichlorosilane, acrylamide gel solution, TEMED, and formamide are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: Many companies provide equipment needed for sequencing experiments; a list of suppliers is provided in CPMB Table 97.80.4711.
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Literature Cited

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