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Probe labelling


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#1 anonymous

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Posted 19 June 2001 - 09:00 PM

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?

#2 anonymous

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Posted 20 July 2001 - 09:00 PM

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!!!




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