Notes For Nucleic Acid Hybridisations Using The Thermo Range Of Equipment; Placing Membranes In A Bottle - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

The final wash is a high stringency wash. Use of a hand held monitor to give an
indication of the decrease in radioactivity as the washes progress is recommended
and will determine if the final wash should be carried out.
7. Wrap the membrane in Saran Wrap and autoradiograph at -70°C in a cassette
with an intensifying screen or use a phosphoimager. Expose initially for 12 hours
(or overnight).
8. If the membrane is to be reprobed, at no stage should the membrane be allowed to
dry out? Removal of the probe on the membrane may be carried out by washing
the membrane for 1-2 hours at 65°C in 5mM Tris HCl pH8.0, 0.2mM EDTA, 0.1 x
Denhardt's reagent.
Notes for Nucleic Acid Hybridisations using the Thermo Range of
Equipment
The Hybridisation Oven range has been designed to provide the optimum conditions for
performing all types of Hybridisation and stringency washing procedures safely and
simply. Hybridisations are performed in bottles to maximise user safety and to minimise
probe volumes.

Placing Membranes in a Bottle

1. Place the bottles to be used in the Oven during the warm up period (approximately
1 hour).
2. Select a piece of support mesh appropriate for the size of the membrane. The
recommended Hybridisation mesh is supplied by Thermo.
3. Pre-wet the mesh and Hybridisation membrane in a suitable tray containing 2 x
SSPE (SSC) (see Figure 3.1).
4. Ensuring that the Hybridisation membrane exactly overlays the mesh, roll both up
into a tight roll.
If more than one membrane is to be hybridised in a bottle, simply overlay further
meshes and membranes as required before rolling. It is important that each
membrane is separated from any other by a piece of mesh. Up to five 20 x 20mm
membranes can be hybridised in a single Hybridisation bottle.
© Thermo Scientific, May 2003. Issue 7
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