PCR DIG-labeling Southern probes

Dickens Lum lumdicks at netvigator.com
Wed Aug 6 09:42:35 EST 1997


Hi Netter,

> I have a 514-bp insert in a pUC12, 2,680-bp, plasmid that I use as a
> DNA
> probe for a Southern blot against rabbit genomic DNA.  I have started
> using Boehringer-Mannheim's PCR DIG-labeling synthesis kit for
> 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 think it is better to cut it out, gel purify and then label
it. Although theoretically the probe can be labeled by labelling the
whole plasmid, the efficiency is much lower. Besides, you can not
estimate the amount of specific probe generated if the fragment was not
cut out.

            Hope this help....

Regards,
Lum




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