Probe labelling - (Jun/19/2001 )
I have been trying to label a 190bp fragment (purified by gel extraction) with radioactive CTP using the random priming kit from Amersham. I tried the control DNA first (3.5Kb) and purified the reaction mixture through Chroma-spin TE-100 spin columns from Clontech. It worked fine and the probe was very hot. When I tried the same using the 190bp fragment as template I got very little radiation. Could it be that because the template is so small most of the generated fragments are retained in the column?Is it the template gel extraction?Any ideas?
If I had to guess, it is the clean up step. I use random priming from Biorad and usually throw the entire reaction mixture into the hybridization solution. I get fine results!!!