cleaning up my probes

Tori Brophy torih at
Mon May 27 06:21:57 EST 1996

When we make band shift probes, we use mini spin-columns to remove the 
unincorporated nucleotides.  Pharmacia sells MicroSpin columns that allow 
you to clean up your probe in about 5 minutes.  If you don't want to 
spend the money on them ($120 for 50 columns), you can make your own with 
Sephadex G-25 and 1 ml syringes (it takes a little longer because you 
can't spin them).  The first few times you use your own column, you'll 
have to take fractions and determine when your probe comes off.  We spot 
a little bit on a piece of filter paper and count it on a scintillation 
counter.  This should still be faster than running a gel and isolating 
the DNA.

Feel free to email me if you want more details.

Tori Brophy
torih at
Dept of Genetics
University of Washington
Seattle, WA      

Antonio Izzo wrote:
> In article <uvlpxcUoxsjQ089yn at>, jtardiff at (Jil
> Tardiff) wrote:
> > Jennifer,
> >
> >   I used to do band-shifts with small fragments (I *think* they were 40mers)
> >   and had similar problems.  The best solution I found was to gel-purify
> >   the fragments through acrylamide before running the band-shift assay.
> >   Usually the fragments were hot enough that I could directly expose a sheet
> >   of film for 5-10 minutes (wrapping the acrylamide gel in Saran Wrap) and
> >   use the signal to find the probe.  Then just cut out the band and isolate
> >   using your favorite method. It's a bit time consuming, but it eliminated
> >   the unincorporated nts.
> >
> >   Good Luck,
> >
> >    Jil
> >
> >    /* Jil Tardiff MD, PhD *****************/
> >    /* Albert Einstein College of Medicine**/
> >    /* Columbia-Presbyterian Medical Center*/
> To avoid having to locate the DNA with the radioactive probes you can also
> use a flourescent glass plate. After running the DNA on the gel lay the gel
> on top of the plate on some saran wrap. Shining a UV light on it gives a
> shadow where the DNA is located which allows you to cut the band out
> easily. We elute the DNA out of the gel afterwards by "crushing" the
> cut-out band into little bits (by pushing it through a syringe) and soaking
> it in a  0.5 M ammonium acetate 0.001 M EDTA solution for a day or two.
> This might lessen the hassle of trying to locate the radioactivity?
> hope this helps!
> Antonio Izzo
> Research Associate

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