Reagents and Radioisotopes Used to Manipulate Nucleic Acids

Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.4
DOI:  10.1002/0471142727.mb0304s09
Online Posting Date:  May, 2001
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Abstract

This unit provides recipes and instructions for preparing many common and useful solutions that are used in nucleic acids research. Included are enzyme buffers, reaction conditions for many restriction enzymes, stock solutions of nucleoside triphosphates, as well as numerous procedures for isolating and quantifying the radioactivity of various radioisotopes.

     
 
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Table of Contents

  • Enzymes for Modifying and Radioactively Labeling Nucleic Acids
  • Stock Solutions
  • 10× Enzyme Buffers
  • Enzyme Reaction Conditions and Applications
  • Nucleoside Triphosphates
  • Basic Protocol 1: Measuring Radioactivity in DNA and RNA by Acid Precipitation
  • Basic Protocol 2: Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors by Column Chromatography
  • Alternate Protocol 1: Spin‐Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Radioactivity in DNA and RNA by Acid Precipitation

  Materials
  • 500 µg/ml sonicated salmon sperm DNA in TE buffer
  • Ice‐cold acid precipitation solution ( appendix 22)
  • 100% ethanol
  • Glass microfiber filters (2.4‐cm diameter, Whatman GF/A)
  • Filtration device
  • Heat lamp (optional)
  • Scintillation fluid and vials

Basic Protocol 2: Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors by Column Chromatography

  Materials
  • TE buffer ( appendix 22)
  • 6 1/2‐in. Pasteur pipet
  • Silanized glass wool ( appendix 3A)
  • Swelled column resin (e.g., Sephadex G‐50 or Bio‐Gel P‐60 from Bio‐Rad)

Alternate Protocol 1: Spin‐Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors

  Additional Materials
  • 5‐ml disposable syringe
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Figures

Videos

Literature Cited

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