Chapter 1: Introduction - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

The immobilisation of nucleic acids on a solid support and subsequent detection of
specific sequences of interest is one of the fundamental techniques of modern molecular
biology. A number of different techniques may be used to analyse target sequences from
various sources. These techniques are all variations on a theme, which can be
summarised according to the source of DNA or RNA as follows: -
1. Dot/Slot Blot Hybridisation
A sample of unfractionated DNA or RNA immobilised on a membrane.
2. Colony/Plaque Blot Hybridisation
DNA derived from cultured bacteria directly or from bacteriophage plaques and
immobilised on a membrane.
3. Southern/Northern Blot Hybridisation
DNA or RNA, which has been size fractionated by gel electrophoresis and then
transferred to a membrane.
4. Western Blot Hybridisation
Western blotting refers to the detection of polypeptides blotted on to nitrocellulose
with antibodies, and is outside the scope of this manual.
In each case, the basic principle remains the same. The nucleic acid for analysis is
immobilised on a solid support and then hybridised with a specific, single-stranded,
Hybridisation 'probe' complementary in base sequence to the region of interest, and
labelled to facilitate its detection.
The solid support most widely used for Hybridisation reactions is the nylon Hybridisation
membrane. This offers several advantages over alternative matrices such as
nitrocellulose, most notably high physical strength and binding capacity. The DNA is
immobilised on the support by baking or UV cross-linking.
The Thermo range of Hybridisation equipment offers a complete system for the safest
and most efficient processing of all types of blot. The equipment is invaluable for every
stage of the nucleic acid immobilisation and detection procedure, from the initial
electrophoretic separation, through to vacuum transfer and finally, Hybridisation and
washing. The procedures outlined in this booklet are intended as general guidelines for
the use of Thermo Hybridisation Ovens and may not be ideally suited for every research
application.
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© Thermo Scientific, May 2003. Issue 7
CHAPTER 1
HYBRIDISATION GUIDE
INTRODUCTION
1