Factorsresultinginsignal Lowerthanexpected - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Section 11
Appendix
FactorsResultinginSignal
LowerthanExpected
A-4 Shake 'n Stack
1. Insufficient exposure time of membrane to film during
autoradiography.
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
radiolabelled 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.
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