Summary Of Factors Resulting In High Backgrounds - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Summary of Factors Resulting in High Backgrounds

1. Hybridisation solutions and/or wash solutions not pre-warmed before use.
2. Probe concentrations too high or probe not denatured. When transferring
Hybridisation protocols to bottles the volumes will be reduced. Ensure that probe
concentrations are adjusted accordingly.
3. Unincorporated nucleotides not removed from probe solution.
4. Insufficient prehybridisation or blocking agents in prehybridisation and Hybridisation
solutions (e.g. Denhardt's reagent and salmon sperm DNA). An adequate
prehybridisation is important to block non-specific Hybridisation to the membrane.
5. Hybridisation and/or washing conditions not stringent enough: -
(i) Decrease salt concentration.
(ii) Increase temperature.
(iii) Increase concentration of SDS.
(iv) Increase wash times.
6. Membranes drying out. This may often be the cause of an apparent overlap
problem and may result from: -
(i) Too low a probe volume.
(ii) Too slow a change over of solutions, particularly when bulk processing.
(iii) Oven not level.
(iv) Excessive variable axis angle.
7. Residual agarose on membranes may cause foggy backgrounds. Membranes
should be rinsed in 2 x SSC to remove residual agarose and excess salt after
blotting and prior to fixing (especially following vacuum blotting).
8. Multiple filters not separated by mesh in bottles.
9. Autoradiography problems. Random black spots and "lightening flash" markings on
autoradiographs may be due to static electricity.
© Thermo Scientific, May 2003. Issue 7
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