Inquiry (again) re: DIG DNA detection with B-M kit, Formamide...

TIM CHIPMAN 901106c at axe.acadiau.ca
Wed Jul 21 11:12:46 EST 1993


I am still trying to troubleshoot the DIG DNA detection procedure, and gosh, 
what a great time I am having. :)

I have a question which hopefully others who have done troubleshooting with 
this procedure:

Does anyone have experience with:

(1) Consequences of shaking the membrane while color pcp. develops?
(2) Consequences of using NON-FILTERED SDS and Sarcosine stock solutions?
    (and of course non-autoclaved...)
(3) Consequences of using 50% Formamide prehyb/hyb solutions that are made up
    of formamide that has NOT been de-ionized with resin beads?

These are all holes in my procedure that have come up; and I am somewhat 
curious if they might explain my troubles, which consist of low sensitivity (
ie: no signal from all but control DNA lanes), and VERY high background 
levels (ie: entire membrane a mottled deep blue colour)

If you can help, I would GREATLY appreciate it if you could mail advice to 
me.

My email address is 901106c at axe.acadiau.ca if you can't autoreply to this 
posting.

Thanks!

Tim Chipman



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