Chapter 7: Troubleshooting Guide For Nucleic Acid; Background Reduction - General; Prehybridisation Procedure - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

CHAPTER 7
HYBRIDISATION GUIDE
TROUBLESHOOTING GUIDE FOR NUCLEIC ACID HYBRIDISATIONS
Our bottle system is intrinsically simpler and safer to use than other methods of
Hybridisation such as Hybridisation in bags or plastic boxes. In Hybridisation Ovens, the
temperature of the solutions is precisely controlled and regulated and, in addition, the
rotisserie device ensures that the solutions are continuously and evenly distributed over
the membrane. Thus, the optimum conditions for Hybridisation and washing are
maintained throughout when using this system. However, during the transfer of protocols
from bags to bottles some minor adjustments to the protocols may be necessary.
When loading the mesh and membranes into the bottles, air bubbles should be avoided.
Ensure that the Oven is sited on a flat surface so that the probe solution is distributed
evenly along the length of the bottles and that there is sufficient probe solution to cover
the entire membrane. On occasions the mesh and membrane can become tightly rolled
up in the bottle. This occurs if the mesh is loaded incorrectly (see Figure 3 in Chapter 3).

Background Reduction - General

All solutions for nucleic acid Hybridisations should be prepared using distilled water and
highest quality reagents in clean glassware. In particular, water with a high organic
content will cause bad background problems. Formamide should be freshly de-ionised.
Membranes should always be handled wearing gloves or with forceps. The following
should also be taken into consideration: -

Prehybridisation Procedure

Prehybridisation is required to block the sites on the nylon membrane, which the probe
would otherwise bind to non-specifically. Failure to carry out adequate preHybridisation
results in high backgrounds. If dextran sulphate is used in the Hybridisation solution, then
it must also be included in the prehybridisation solution.
© Thermo Scientific, May 2003. Issue 7
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