probe cleaning

futers at bpxtal.leeds.ac.uk futers at bpxtal.leeds.ac.uk
Tue Mar 29 04:27:14 EST 1994


In article <sticknbd-280394154449 at 129.59.16.44>,
 sticknbd at miranda.cc.vanderbilt.edu (B Stickney) writes:
>I use Boehringer Mannheim's random primed kit to make DNA probes
>for Northerns.  The kit says a Sephadex G-50 column is not necessary
>to clean up the probe.  However, I don't see how you'd properly evaluate
>your probe in a scintillation counter without running it through a column.
>Also, what would be the consequence of adding a bunch of unincorporated
>32P-labelled dCTPs to a blot?  It seems to me G-50 or something like it
>absolutely is necessary.  Any ideas?
>sticknbd at miranda.cc.vanderibilt.edu
followed their instructions and after the labelling reaction, just heat
denatured the probe and added it to the hybridisation solution.  No
removal of unincorporated dCTP.  The backgrounds were clean.  Therefore at
least for Southern blots I do not think it neccessery to clean the probe
and if you really need to measure the incorporation you can TCA ppt an
aliquot for counting.  BTW the Megaprime kit uses nonamers rather than
hexamers which might make it less important to clean the probe.

           Simon Futers (futers at biovax.leeds.ac.uk)





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