Calculating Tm for mismatch on Northern

Anton Scott Goustin asg at cmb.biosci.wayne.edu
Thu Jul 27 20:56:41 EST 1995


neale at mbcf.stjude.org writes:
>We have a partial cDNA clone from mouse that we wish to screen *human* northern blots. So far, under our usual stringency (50% formamide, 6xSSC, 1% SDS, 42 C) of hybridization we see no signal to very weak signal in the human samples.
>
>My question is whether you can calculate a lower stringency and wash of
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these blots the same as you would for Southerns? My first instinct is that I can, but maybe I overlooking something.

I don't understand the question.  First, WASH conditions won't bring out a signal that isn't there from hybridization.  You must lower the formamide, for example to 32% or 40%.  Calculations are useful only if you have the sequences for both, and you know the regions covered by your probe.  You will be just as happy if you do it empirically, hybridizing at a guess %, for example 35% formamide.  If you are on the low side, your problem will be noise, but you will have signal.  You can often get rid of noise in stringent washes (just don't let the blots ever dry out completely).




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