probes for southern blot - molecular genetics (Mar/08/2006 )
i am supposed to do southern hybridization.please tell me how to make probes for it.
Typically probes are 100-200 bp long and created by PCR of your sequence of interest and then end labelled with radio-isotopes. However, I would recommend that you get a DIG kit from Roche, they have excellent protocols for southerns etc, and provide great results. It also saves using radio-isotopes. With DIG you can label by end labelling, nucleotide replacement or create probes directly by PCR.
If you need to use isotopes then you will probably end label your probe using a terminal transferase, which should come with a protocol when you buy it.
I would also recommend the DIG kit from Roche for probes the size of 100-500bp. However I had problems creating probes above this size.