Summary Of Factors Resulting In A Signal Lower Than Expected - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Summary of Factors Resulting in a Signal Lower than Expected

1. Insufficient exposure time of membrane to film during autoradiography or
phosphoimaging.
2. Inefficient transfer and/or binding of nucleic acids to the nylon membrane.
3. Target sequence present at very low copy numbers. Increase the amount of sample
loaded on to the gel.
4. Probe sequence not present in sufficient quantities. Increase the concentration of
probe or include 10% dextran sulphate, which reduces the solvent volume and has
the same effect.
5. No probe homology.
6. Double-stranded DNA probe was not denatured - see standard protocols.
Alternatively, probe degraded. This is more likely to occur when using RNA probes.
7. The specific activity of the probe was too low. Consider factors such as the probe
concentration during the labelling reaction, half-life of radio-labelled triphosphates,
etc.
8. Hybridisation and/or washing conditions were too stringent: -
(i) Increase salt concentration.
(ii) Decrease temperature.
(iii) Reduce concentration of SDS.
(iv) Reduce wash times.
9. The Hybridisation time was too short.
© Thermo Scientific, May 2003. Issue 7
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