Digestion of DNA with Restriction Endonucleases

Kenneth D. Bloch1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.8
DOI:  10.1002/0471142735.im1008s02
Online Posting Date:  May, 2001
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Abstract

Restriction endonucleases recognize short DNA sequences and catalyze the cleavage of double‐stranded DNA at specific sites within or adjacent to the recognition sequences. This unit describes how to cleave DNA and refers the investigator to frequently updated manuals by endonuclease suppliers for the precise optimized conditions. Alternate procedures are given for digesting a DNA sample with several different enzymes and digesting multiple DNA samples with the same endonuclease.

     
 
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Table of Contents

  • Basic Protocol 1: Standard Cleavage Reaction
  • Alternate Protocol 1: Cleavage with Multiple Restriction Endonucleases
  • Alternate Protocol 2: Cleavage of Multiple DNA Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Standard Cleavage Reaction

  Materials
  • DNA sample: 0.1 to 4 µg DNA in H 2O or TE buffer
  • recipe10× restriction endonuclease buffer
  • Restriction endonuclease
  • Loading buffer (unit 10.4)
  • 0.5 M EDTA (optional)
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Figures

Videos

Literature Cited

Literature Cited
  American Chemical Society. 1991. Biotech Buyers' Guide 1991. ACS, Washington, D.C.
   Fuchs, R. and Blakesley, R. 1983. Guide to the use of type II restriction endonucleases. Methods Enzymol. 100:3‐38.
   McClelland, M., Hanish, J., Nelson, M., and Patel, Y. 1988. KGB: A single buffer for all restriction endonucleases. Nucl. Acids Res. 16:364.
   O'Farrell, P.H., Kutter, E., Nakanishe, M. 1980. Mol. & Gen. Genet. 179:411‐435.
   Roberts, R.J. and Macelis, D. 1991. Restriction enzymes and their isoschizomers. Nucl. Acids Res. 19(Supp.#1):2077‐2109.
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