southern blot - southern blot- restriction of genomic DNA (Feb/04/2008 )

hi all!
this is a really stupid question, but il ask it anyway as i am very confused!
how do you know what enzme to digest your gDNA for a southern blot with? i just dont understand the principle.
can anyone help me please?

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Genomic DNA is very large so if you didn't cut it you wouldn't see any discrete bands on your gel. It's best to use something that will cut often, but not too often. Also its best to use cheap enzymes as what's the point of using something expensive if you don't have to? If you know exactly what you are trying to detect and your region has already been sequenced, then you can certainly use a restriction digest program to help you decide which enzyme would be best to help you see what you want.

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You should select an enzyme in which you know where it cuts in your region of interest and there fore you know what size to expect.

So if you are genotyping, you will get a band of known size (based on where it cuts in your sequence, which you know) and then bands based on where its at in the genome... copy number and number of distinct insertions complicate things.


But yes, the short answer is gDNA is very big and you need to fragment it to see anything on a gel.

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