Chapter 3: Hybridisation Procedures; Southern Blot Dna Hybridisations - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Nucleic acid hybridisations are conveniently performed in the dedicated Hybridisation
equipment available from Thermo.
The Hybridisation Oven range consists of incubators with integral rotisserie devices,
which can accommodate 4, 10 or 14 Hybridisation bottles, 35mm in diameter, in order to
perform Hybridisations in minimal probe volumes with fluid moving continually over the
membrane.

Southern Blot DNA Hybridisations

The following protocol is broadly applicable to Hybridisations using DNA probes of 50bp
and above, following transfer and fixing of DNA to membranes as described in Chapter
2. For Hybridisations using oligonucleotides refer to Chapter 6. The Hybridisation
procedure consists of four stages: -
1. Prehybridisation
2. Hybridisation
3. Stringency washing
4. Autoradiography
For detailed notes on Hybridisation specific to Thermo equipment, refer to 'Notes for
Nucleic Acid Hybridisation' on page 11.
1. Prehybridisation is carried out by incubating the membrane in Southern Blot
Prehybridisation Buffer (for recipe see page 30). Denature salmon sperm DNA
by boiling for 5 minutes and then chilling on ice. Add the denatured salmon sperm
DNA to the buffer to a final concentration of 50µg/ml.
2. Incubate with agitation or in a rotisserie for a least 1-hour at 65°C.
3. The volume of prehybridisation buffer required varies according to the Hybridisation
system being utilised. In general terms, the minimum volume of buffer should be
used such that the membrane is covered by the fluid at all times (approximately
2
0.1ml/cm ), or if in Hybridisation bottles, 10ml for a large bottle and 5ml for a small
bottle.
© Thermo Scientific, May 2003. Issue 7
CHAPTER 3
HYBRIDISATION GUIDE
HYBRIDISATION PROCEDURES
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