minimum probe size

Caroline Szymeczek-Seay css at med.unc.edu
Sun Sep 26 10:34:29 EST 1999


Hi Kev,
I don't know the minimum size template for random priming, although I've
successfully probed a northern blot with a random primed probe made from
a 250 or 300 bp template.  The problem is that the smaller the template,
the more small probe fragments you get, and more background, and less
signal.  For a 33 base oligo, I would recommend that you have the oligo
synthesized with fluorescein "built in."  Life Technologies (no
affliliation) and I'm sure lots of other companies do this.  You
probably could also, instead of random priming, use a 6-mer or an 8-mer
specific to your oligo and "specific prime" it.  I haven't tried this,
but perhaps others may comment.  And you could kinase it, unless you're
avoiding radioactivity.  I've done biotin labelling of oligos using
terminal deoxytransferase....that's a very standard methodology. 
Anyway, my advice, and I'm sure others will give you other advise, is to
*not* try the random priming thing. There are lots of other methods,
unless you have some specific reason for wanting to do it the way you're
proposing.  Good luck...

caroline

"K.A. Floyd" wrote:
> 
> Hi,
> 
> Sorry for a basic question.
> 
> What is the is the smallest size I can get away making a random
> prime labelled probe with the Amersham Random-Prime labelling system used
> in ECL.  I couldn't (i.e probably missed) find anything in their
> literature.
> 
> Could I get away with labelling a 33nt Oligo with fluroscein with this
> module??
> 
> regards
> 
> Kev
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