HELP! Desperately need a dot blot method

Wendy Ingram w.ingram at cmcb.REMOVE-THIS-BIT.uq.edu.au
Tue Jun 30 02:07:48 EST 1998


Hi Cathy,

I may have a simple solution to your problem.  When I do colony lifts of
E.coli containing plasmids of interest I use the "Turbo Juice" method. 
Basically this involves using dry membrane to lift the colonies.  The
membrane is then put DNA side up on a bit of 3M paper that has been
soaked with Turbo Juice (simply 2x SSC + 5% SDS) and popped in the
microwave on high for 3-4 minutes until very hot and dry.  The membrane
can then be hybridised straight away; no washing or UV linking required,
just pop straight into prehyb then hyb mix.

Anyway, often when I do lifts like this I just spot my positive and
negative control plasmids on an offcut of dry membrane and pop then
through the same pocedure as above after the spots have dried a few
minutes.  I know it seams to easy to be true but it always works. No
reason why it should not work for a whole blot of spotted plasmids.  

A word of Warning though: You can only do this with nylon membrane,
don't microwave nitrocellulose!

Please let me know if you need any more info or references.

Good Luck,

Wendy Ingram
CMCB, University of Queensland
Australia


>>>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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