HELP! Desperately need a dot blot method

user at ichr.uwa.edu.au user at ichr.uwa.edu.au
Mon Jun 29 21:39:53 EST 1998


I'm trying to do a dot blot of a 96-well tray containing supercoiled
plasmid. I can't find a method that doesn't involve denaturation by
boiling for 10 mins. I'm trying to avoid this as it would be awkward with
96 samples. 

I've tried blotting (10pg of positive control) onto to a (dry) neutral
membrane, placing the membranes on 0.4M NaOH soaked 3MM Whatmans for
10mins., neutralising with 1M Tris (pH 7.5) and UV crosslinking. I then
washed with 2 x SSC before hybridising. I was thinking that maybe
microwaving after the NaOH step might help to denature the DNA. 

Would love some feedback if anyone has tried this before and has a tried
and true method or has any constructive advice.

Cathy Fussell
Perth, Western Australia

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TVW Telethon Institute for Child Health Research
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Western Australia

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