I have a question regarding the hybridisation step in Southern blotting. I understand that once the probes are added, the membrane is washed in buffer of increasing stringency to increase the specificity of probe binding.
Why a low-stringency buffer is used before a high-stringency buffer? Or conversely, what would be the consequences of washing the membrane with only high-stringency buffer?
I tried looking around online for an answer, but I struggle to find anything.
Thanks!
I think that doing a low stringency wash first is very safe and washes away all wetting reaction mix and low stringency binding without weakening the high stringency high specificity binding signal. If we just went straight in with high stringency wash by the time that we have washed away the non-bound but nylon wetting reaction mix we will also have washed off some of the high specificity signal so made it a weaker signal so we should be able to rinse the membrane 2 times at high salt without any effect on the specific signal.
In the setup stages of blotting it is risky to wash high stringency first because the stringency might be too high and all of the specific signal washes off but starting with low sringency we get a signal against a high background which tells you it has worked and then the stringent washes improve the signal:noise ratio and wash off more of the low specificity bound probe
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