Thermo Scientific Shake 'n Stack 6244, Operating And Maintenance Manual

Page: 45 / 92

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 pre-hybridisation or blocking agents in pre-hybridisation
and hybridisation solutions (e.g. Denhardt’s reagent and salmon sperm
DNA). An adequate pre-hybridisation 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.
Shake ‘n Stack A-3 Thermo Scientific
Section 11
Appendix
Factors Resulting in
High Backgrounds