Increasing probe sensitivity

Paul Taylor p.taylor7 at pgrad.unimelb.edu.au
Sat Jul 1 22:46:50 EST 2000


Hi All, 

I am trying to detect down to 10e4 copies of a gene. At the moment I can
only get down to 10e6 copies. The extra level of sensitivity is necessary. I
am using a random priming probe synthesis and commercial hyb buffers to make
things happen more quickly. The probe is about 400bp long and has a G+C
content of 60%. All membranes are prepared by slot blotting. Can anyone
offer any neat tricks for increasing sensitivity of hybridisations? Today I
am trying increased probe template concentration in the labelling reaction
to try and rule out probe exhaustion - that is, I want to be sure that all
the probe is not being snaffled by the higher target concentrations.....

Thanks,

Paul


Paul Taylor
Microbial Ecology and Bioremediation Laboratory
Department of Microbiology and Immunology
The University of Melbourne
Victoria 3010

p    +61 3 8344 5706
f    +61 3 9347 1540
e    p.taylor7 at pgrad.unimelb.edu.au


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