Long transcript Northerns

Walter Blank wblank01 at tufts.edu
Mon Jul 23 12:58:19 EST 2001


Hi all, 

What is the longest RNA molecule one could reasonably expect to detect on a
Northern blot?  I'm looking for a ~20 kb bacterial transcript after
separation on a .8% agarose formaldehyde gel.  My northern shows a band
immediately below the well (looks like it barely entered the gel) with a
small amount of smearing well above the 9kb NEB RNA marker.  I used Qiagen's
RNeasy mini kit without DNAse for the preps but I'm pretty sure that it's
not DNA that I'm detecting (a mutant with the operon disrupted doesn't show
the high MW band but does have considerable smearing further down)
My questions:
1) Will a transcript that size migrate normally in a gel? (My guess is that
it ought to)
2) If it will migrate normally, what am I seeing at the tops of my lanes?
The denaturing gel conditions should take care of any large complexes,
shouldn't they?
3) Are any larger RNA size markers commercially available?  The longest I've
been able to find is 9kb.  Alternatively, does ssDNA migrate similarly to
RNA?  I'm thinking of just denaturing some lambda HindIII and running that
alongside.

Thanks for any help/comments.
Walter




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