Southern Hybridization - about the probe and DNA concentration (Jun/14/2005 )
It will be great if somebody here can enlighten me. Usually what is the concentration for the probe and DNA to hybridize??? (How many ng) I will not bother the minimum concentration of them. I just want to know whether my probe can successfully hybridize to the target DNA.
For southern blot you only need 5ug of DNA, this is enought, if it was a northen then you need more, around 20 and 30ug of RNA. About the amount of probe you have to use 50ng.
Upps!! Remember that when you are doing a southern blot you need a diefernt % of formamide than in northern blot, 50 and 20.
I use 10ug DNA across the board.
For probe: I random label (Amersham RediPrime II works well) 25ng PCR product or purified plasmid fragment per protocol. Size range 200-700bp preferred.
Probe blot with 2-3E5cpm/ml
If i want to do reverse dot blot, any one have any idea bout the DNA and probe concentration??