HELP! Desperately need a dot blot method
w.ingram at cmcb.REMOVE-THIS-BIT.uq.edu.au
Tue Jun 30 02:07:48 EST 1998
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.
CMCB, University of Queensland
>>>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
>>>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.
>>>Perth, Western Australia
>>>TVW Telethon Institute for Child Health Research
>>>(Company Limited by Guarantee)
>>>Fax 61 9 388 3414
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