Protocol

Preparation of Denaturing Polyacrylamide Gels

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

In 1978, Fred Sanger and Alan Coulson devised a method to pour and run thin polyacrylamide gels, which are now used ubiquitously to resolve the products of DNA sequencing reactions.

MATERIALS

10x TBE electrophoresis buffer

TBE is used at a working strength of 1x (89 mM Tris-borate, 2 mM EDTA) for polyacrylamide gel electrophoresis.

Acrylamide solution (45% w/v)

Ammonium persulfate (1.6% w/v) in H₂O

Deionized H₂O

Detergent, household dishwashing

Ethanol

Optional, please see Step 5.

KOH/Methanol solution

Silanizing fluid

The traditional silanizing fluids (e.g., Sigmacote from Sigma and Repelcote from BDH Inc.) contain dichlorodimethylsilane, which is toxic, volatile, and . . . [Full Text of this Article]

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