cleaning up my probes

Tori Brophy torih at genetics.washington.edu
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 genetics.washington.edu
Dept of Genetics
University of Washington
Seattle, WA      


Antonio Izzo wrote:
> 
> In article <uvlpxcUoxsjQ089yn at westnet.com>, jtardiff at westnet.com (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
> http://mmr.bmb.colostate.edu



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