Reagents and Radioisotopes Used to Manipulate Nucleic Acids

Kevin Struhl1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.9
DOI:  10.1002/0471142735.im1009s02
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The application of molecular biological techniques to immunological studies is based in large part on the manipulation of nucleic acids‐‐RNA, DNA, and oligonucleotides. This unit offers very basic procedures that are used in a number of protocols later in the chapter. In particular, preparation of nucleoside triphosphates‐‐the substrates for almost all synthetic reactions using nucleic acids‐‐is described; the relevant radioisotopes, 32P, 35S, and 3H are discussed; and methods for quantitating radioisotope and biotin incorporation into nucleic acids are presented. Finally, column chromatographic (gel filtration) procedures for separating labeled DNA from synthetic precursors are described.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Preparation of Nucleoside Triphosphates
  • Radioisotopes for Labeling Nucleic Acids
  • Basic Protocol 2: Quantitation of Radioactivity in DNA and RNA by Acid Precipitation
  • Basic Protocol 3: Gel‐Filtration Procedure for Separating Radiolabeled DNA from Unincorporated dNTP Precursors
  • Alternate Protocol 1: Spin‐Column Procedure for Separating Radiolabeled DNA from Unincorporated dNTP Precursors
  • 10× Enzyme Buffers
  • Reagents and Solutions
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Nucleoside Triphosphates

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

Basic Protocol 2: Quantitation of Radioactivity in DNA and RNA by Acid Precipitation

  Materials
  • TE buffer ( appendix 2A)
  • 6 ½‐in. (16.5‐cm) Pasteur pipet
  • Silanized glass wool ( appendix 3A)
  • Equilibrated column resin (e.g., Sephadex G‐50 from Pharmacia LKB or Bio‐Gel P‐60 from Bio‐Rad)

Basic Protocol 3: Gel‐Filtration Procedure for Separating Radiolabeled DNA from Unincorporated dNTP Precursors

  Additional Materials
  • 5‐ml disposable syringe
  • 15‐ml polypropylene centrifuge tube
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library