Dot and Slot Blotting of DNA

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.9B
DOI:  10.1002/0471142727.mb0209bs21
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Dot and slot blotting are simple techniques for immobilizing bulk unfractionated DNA on a nitrocellulose or nylon membrane. Hybridization analysis can then be carried out to determine the relative abundance of target sequences in the blotted DNA preparations. Dot and slot blots differ only in the geometry of the blot, a series of spots giving a hybridization pattern that is amenable to analysis by densitometric scanning. Samples are usually applied to the membrane using a manifold attached to a suction device. The describes such a procedure for dot or slot blotting on an uncharged nylon membrane; annotations to the steps detail the minor modifications that are needed if blotting onto nitrocellulose. The first alternate protocol describes the more major changes required for blotting with a positively charged nylon membrane. A second alternate protocol describes preparation of dot blots by spotting the samples onto the membrane by hand.

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

Table of Contents

  • Basic Protocol 1: Dot and Slot Blotting of DNA onto Uncharged Nylon and Nitrocellulose Membranes Using a Manifold
  • Alternate Protocol 1: Dot and Slot Blotting of DNA onto a Positively Charged Nylon Membrane Using a Manifold
  • Alternate Protocol 2: Manual Preparation of a DNA Dot Blot
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Dot and Slot Blotting of DNA onto Uncharged Nylon and Nitrocellulose Membranes Using a Manifold

  Materials
  • 6× and 20× SSC ( appendix 22)
  • DNA samples to be analyzed
  • Denaturation solution: 1.5 M NaCl/0.5 M NaOH (store at room temperature)
  • Neutralization solution: 1 M NaCl/0.5 M Tris⋅Cl, pH 7.0 (store at room temperature)
  • Uncharged nylon or nitrocellulose membrane (see Table 97.80.4711, unit 2.9, for suppliers)
  • Whatman 3MM filter paper sheets
  • Dot/slot blotting manifold (e.g., Bio‐Rad Bio‐Dot SF or Schleicher and Schuell Minifold II)
  • UV‐transparent plastic wrap (e.g., Saran Wrap)
  • UV transilluminator (unit 2.5) for nylon membranes

Alternate Protocol 1: Dot and Slot Blotting of DNA onto a Positively Charged Nylon Membrane Using a Manifold

  Additional Materials
  • Positively charged nylon membrane (see Table 97.80.4711, unit 2.9, for suppliers)
  • 0.4 M and 1 M NaOH ( appendix 22)
  • 200 mM EDTA, pH 8.2 ( appendix 22)
  • 2× SSC ( appendix 22)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Res. 7:1541‐1552.
Key Reference
   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 at Oxford University Press, Oxford.
  Describes dot and slot blotting in some detail.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library