Appendix I Troubleshooting Guide For Nucleic Acid Hybridisations; Background Reduction - General - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Troubleshooting Guide for
Appendix I
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
the Hybridisation Ovens, the temperature of the solutions is precisely
controlled and regulated, and, 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 positioned 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 occasion, the mesh and membrane can become tightly rolled up in the
bottle. This occurs if the mesh is loaded incorrectly (see Figure 4-1 in
Section 4).
Background
All solutions for nucleic acid hybridisations should be prepared using
distilled water and highest quality reagents in clean glassware. In particular,
Reduction - General
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 considerations
should also be applied:
Pre-Hybridisation Procedure
Pre-hybridisation is required to block the sites on the nylon membrane,
which the probe would otherwise bind to non-specifically. Failure to carry
out adequate pre-hybridisation results in high backgrounds. If dextran
sulphate is used in the hybridisation solution, then it must also be
included in the pre-hybridisation solution.
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