Hydroxylamine-Osmium tetroxide help

Tue Feb 21 14:18:42 EST 1995

Hi I am trying to use H.O.T to look at chemical mismatches in 
hybridized DNA.  So far I am trying to see if the chemical cleavage 
will work using nonradioactive DNA.  I follow the protocol as 
outlined in the "PCR, a practical approach" but start with 1 
microgram of test DNA hybridised with 0.1 to o.5 micrograms of 
"probe" DNA so I can view the results with ethidium bromide staining.  
All I see is a faint smear from the well down to where the band of 
uncut DNA would have been located.  I have tried decreasing the time 
that the hybrid DNA is exposed to the chemicals from 2 hours to 10 
min, with no improvement.

Is anyone else using this technique with PCR-amplified probes?  The 
labeling aspect is another problem that I will approach after I can 
confirm that the chemical cleavage works.

Finally, does anyone have an email address for someone in David 
Bentley's lab in Guy's Hospital, London?  They use this technique 
with end-labeled probes. 

OR an address for someone in the Oliver Miller Lab at the Murdoch Institute in the Royal Children's Hospital, 
Parkville, Victoria, Australia.  They are using this technique with 
radioactive PCR probes.

Thank you, Mo Small

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