Hybridization Analysis of DNA Blots

Terry Brown1, Kevin Struhl2

1 University of Manchester Institute of Science and Technology, Manchester, 2 Harvard Medical School, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 4H
DOI:  10.1002/0471140864.psa04hs13
Online Posting Date:  May, 2001
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Abstract

Restriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. This appendix describes restriction endonucleases and their properties.

     
 
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Table of Contents

  • Basic Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled DNA Probe
  • Alternate Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled RNA Probe
  • Support Protocol 1: Removal of Probes from Hybridized Membranes
  • Support Protocol 2: Preparation of Denatured Salmon Sperm DNA
  • Support Protocol 3: Spin‐Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors
  • Reagents and Solutions
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled DNA Probe

  Materials
  • Probe DNA labeled to a specific activity >1 × 108 dpm/µg
  • Aqueous prehybridization/hybridization (APH) solution: 5× SSC/ recipe5× Denhardt's solution (see recipe)/1% (w/v) SDS/100 µg/ml denatured salmon sperm DNA (see protocol 4; added just before use); room temperature and 68°C
  • 2× SSC/0.1% (w/v) SDS
  • 0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
  • 0.1× SSC/0.1% (w/v) SDS, 68°C
  • 2× and 6× SSC ( appendix 2E)
  • Hybridization oven (e.g., Hybridiser HB‐1, Techne) or 68°C water bath or incubator
  • Hybridization tube or sealable bag and heat sealer

Alternate Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled RNA Probe

  • TE buffer, pH 8.0 ( appendix 2E)
  • Labeling buffer: 200 mM Tris⋅Cl (pH 7.5)/30 mM MgCl 2/10 mM spermidine
  • Nucleotide mix: 2.5 mM ATP/2.5 mM CTP/2.5 mM MGTP/ 20 mM Tris⋅Cl, pH 7.5 (store at −20°C)
  • 200 mM dithiothreitol (DTT), freshly prepared
  • 20 U/µl human placental ribonuclease inhibitor
  • [α‐32P]UTP, 20 mCi/ml (800 Ci/mmol) or 10 mCi/ml (400 Ci/mmol)
  • SP6 or T7 RNA polymerase
  • RNase‐free DNase I
  • 0.25 M EDTA, pH 8.0 ( appendix 2E)
  • Formamide prehybridization/hybridization (FPH) solution: 5× SSC/ 5× Denhardt's solution/50% (w/v) formamide/1% (w/v) SDS/100 µg/ml denatured salmon sperm DNA (see protocol 3; add just before use)
  • 2× SSC containing 25 µg/ml RNase A + 10 U/ml RNase T1

Support Protocol 1: Removal of Probes from Hybridized Membranes

  • Mild stripping solution: 5mM Tris⋅Cl, pH 8.0/0.2 mM EDTA/0.1× Denhardt's solution
  • Moderate stripping solution: 200 mM Tris⋅Cl, pH 8.0/0.1× SSC/0.1% (w/v) SDS
  • 0.4 M NaOH
  • 0.1% (w/v) SDS, 100°C
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Figures

Videos

Literature Cited

Literature Cited
   Brown, T.A., ed. 1991. Molecular Biology Labfax. BIOS Scientific Publishers, Oxford.
   Dyson, N.J. 1991. Immobilization of nucleic acids and hybridization analysis. In Essential Molecular Biology: A Practical Approach, vol. 2 (T.A. Brown, ed.) pp. 111‐156. IRL Press, Oxford.
   Little, P.F.R. and Jackson, I.J. 1987. Application of plasmids containing promoters specific for phage‐encoded RNA polymerases. In DNA Cloning: A Practical Approach, Vol. 3 (D.M. Glover, ed.) pp. 1‐18. IRL Press at Oxford University Press, Oxford.
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