HELP! Desperately need a dot blot method
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Tue Jul 7 02:37:49 EST 1998
We´ve just done such blotting with samples from a 96-well tray. What we did
was to mix DNA (in this case 5ul of a miniprep) with 300ul of 0.5M NaOH, 1.5M
NaCl (denaturation buffer using in library screening; actually we used this
buffer because it was available in the lab. I guess that any alkaline
denaturation will work equally well). Then, we transferred this solution into
a "convertible" dot blot apparatus with its 96-well, 6mm top plate (Life
Technologies, no affilitation), and slowly blotted the samples onto a nylon
membrane. Of course, any dot blot apparatus will work. After blotting, we
added 300ul of denaturation buffer to the wells for washing. The membrane was
removed, briefly rinsed in lots of TE, then air-dried, and UV crosslinked.
Hybridization worked like a charm.
Hope this helps.
user at ichr.uwa.edu.au wrote:
> 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
> TVW Telethon Institute for Child Health Research
> (Company Limited by Guarantee)
> Western Australia
> Fax 61 9 388 3414
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