PCR DIG-labeling Southern probes

Eugenie L. Harris "jean.harris" at stonebow.otago.ac.nz
Tue Aug 5 08:08:32 EST 1997


I used PCR and a purified phage library to DIG-label probes for
Southerns.  It worked OK except when I made the mistake of using Pfu, a
polymerase with 3' exonuclease proofreading activity.  The Pfu corrected
the probe to smitherns and I ended up with nothing.  But I decided to
forget DIG-labeling by PCR since it was in the end more efficient to
amplify up the fragment I wanted to label, run it down a gel, purify the
band with a glass-binding method then DIG label using random priming. 
This may sound more inefficient, but I found I could make enough probe
in one random prime labeling reaction for all future needs whereas I
needed to keep going back to produce more DIG-labeled probe when I
labeled PCR.  If you don't need a lot of probe DIG-labeling by PCR might
be the way to go.

But any way you cut it I think chemiluminescence beats radioisotopes
hands down.

Good luck



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