I am studying about southern hybridization now and I've a doubt.After the DNA has been fragmented using restriction enzymes and obtained on nitrocellulose blot , it is still double stranded (the fragments) so how do single stranded probes bind to it ?
The double-stranded DNA fragments are fractionated by size during the agarose gel electrophoresis. Prior to the so-called “blotting” step, the gel is briefly soaked in a solution of NaOH, which has a relatively high pH. Under these conditions, double-stranded DNA will denature, or “melt” into two single strands. This is the material that is “wicked” or “blotted” onto the nitrocellulose membrane in the presence of monovalent cations.
If the labeled probe DNA is also double-stranded then it is advisable to denture it as well prior to the hybridization step. We used to use boiling hot water to denature the probes.