Probe labelling by PCR
smori at nmsu.edu
Fri Nov 24 23:40:24 EST 1995
Thomas Urbig (Thomas at hastingslab.harvard.edu) wrote:
: Has onybody experience labeling gene probes by PCR and can provide
: reaction conditions for this. It should also be possible to make single
: stranded probes. If so, do you use a single stranded PCR product as
: template or is a standard (double strand) PCR product as template fine.
: Is the lower concentration of the 32P nucleotide a problem or do I have
: to add cold nucleotide?
: Any advice is welcome, thanks in advance
It works but you have to make sure that the conc. of your starting DNA
isn't that great. Then you may still have too many cold copies that
might interfere with your probe.
I like random oligo-labelling ( and actually end labelling I think would be
best) for 2 reasons:
1)In the PCR labelling you'll have such a hot probe that it won't last
you long and you pretty much get to use it once or maybe twice. With
oligo and even better end-labelling -> Much more stable probe. You can use
it for 1 week after making it.... It's all your preference.
2) Oligolabelling and end-labellings are pretty easy.
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
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