2 replies to this topic

[biobunny]

hello everyone~

I have been using the Roche Dig-Filter Hybridization Kit for my southern blotting.

And I realized I am not very clear about the pricinples in the whole procedure.

My first question is for the Prehybridization buffer, if I choose to prepare it myself, then I need the Blocking Reagent meantioned by the Manual, which is a purified fraction of milk powder, reconstituted and diluted in maleic acid buffer. So I wonder why it is not the salmon sperm but milk powder to work in the prehybridization procedure??

My second question is what is in the Blocking Solution which should be used after hybridization and before adding DIG-antibody?----Some protein? or DIG analog? or Salmon sperm?

Thank you very much if you could be so kind as to answer my questions~

[bob1]

Do you have the Roche DIG manual? The proceedure is explained in detail in there, as well as the components of the various buffers and solutions.

Basically the system works by blocking any available sites on the membrane so that your probe will not be able to just bind to the membrane, as it would preferentially do if there was no blocking proceedure prior to the hybridisation. The blocking reagent may contain some non-specific DNA sequences, as well as milk powder for this purpose. However, in theory, milk powder should work all by itself, as it will prevent the probe DNA from binding to the membrane by steric hindrance.

[biobunny]

To BOB1, thanks a lot!

I do have the Manual and it says that the prehybridization buffer and the blocking buffer used before adding DIG-antibody are prepared from the same stock blocking buffer as part of the DIG Wash and Block Set. So milk powder works in terms of blocking the membrane area without DNA before hybridizaiotn and also works in terms of blocking both the blank area without sample DNA and the area with sample DNA unbound by the probe??