PCR DIG-labeling Southern probes

bqamar at paknet1.ptc.pk bqamar at paknet1.ptc.pk
Mon Aug 4 22:51:11 EST 1997


In article <19970804183101.OAA20840 at ladder01.news.aol.com>,
  svlusagi2 at aol.com (SVLUsagi2) wrote:
>
> digoxigenin-labeling of this probe.  Is it better for me to cut out the
> 514-bp insert from the plasmid
> (using Pst I), gel purify it, then use this as the DNA template for the
> PCR DIG-labeling reaction, or can I just use the whole plasmid as the DNA
> template?  Thanks in advance for any suggestions.

I do not really see why you have to cut the insert out and label it.  As
long as you have primers that flank the insert you can use those to
directly label your PCR amplimer with DIG.  I once did have a problem in
amplifying an uncut plasmid (I have no idea why) but what I did was nick
it with an enzyme that had only one site in it and I then labeled it
without any problems.

Happy labeling.

Raheel Qamar

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