Radio-labelling EthBr stained DNA

Michael Holloway mhollowa at LIBSERV1.IC.SUNYSB.EDU
Sat Feb 15 21:28:25 EST 1992


> 
> In article <1992Feb10.002954.11436 at minyos.xx.rmit.oz.au>, s860780 at minyos.xx.rmit.oz.au (Clinton Grant) writes:
> > 
> >     I am posting this on behalf of a freind:
> >     Does anybody know whether DNA taken from a stained gel (and GeneCleaned)
> > is OK to label up with P32 (using an oligonucleotide labelling kit)? Does the 
> > EthBr interfere with the labelling reaction?
> > 
> >                     Thanks in advance. 
> -- 
> EtBr does not interfere with labelling.  If you run your DNA on a low melt gel
> like SeaPlaque GTG (FMC), all you need to do is slice out your band and melt
> it.  Then, dilute your melted slice with water so that you know you have
> approximately 50 ng per aliquot, remove an aliquot to label (like 5 ul)
> and add it to the water specified in the reaction protocol.  
> Boil for 5 min, add the random primer, BSA, 32PdNTP, and Klenow polymerase.  
> The results are excellent and you save yourself the trouble and loss of DNA 
> which results from trying to purify the DNA from the gel slice.  We make up all
> of our own reagents for random primed labelling, using the protocol in the 
> original paper by Feinberg and Vogelstein (Analyt. Biochem. 137:266-267, 1984), 
> so we don't have the expense of the labelling kit, either.  However, we have a
> friend in a neighboring lab who likes to use a Pharmacia labelling kit, and she
> routinely uses melted gel slices to label.  She pre-aliquots her melted slice
> so that she only has to go to the freezer and get out a tube to set up probe.
> If you purify the probe away from unincoporated nucleotide, be sure to heat it
> at the same temp that you melted the gel first, so that you can be sure there
> is no semi-solid agarose left in the reaction to interfere with column flow.
> If you would like more information, e-mail to me!
> ------------------------------------------------------------------------------
> Barbara Bugg, M.S.  bugg at mbcf.stjude.org       "I refuse to have a battle of
> St. Jude Children's Research Hospital          wits with an unarmed person."
> Memphis,  TN  USA
> ------------------------------------------------------------------------------
> 
> 
        Ditto.  The Stratagene Prime-It kit has a protocol for LMP agarose
in their instruction sheets.  I've never found it necessary to reheat prior
to the spin column though.  The kit protocol calls for the fragment to be 
diluted with 3 ml diH2O per gram of gel.
        Barbara, doesn't the original Vogelstein protocol use sheared ssDNA?  
Does this work as well as synthetic oligos or does the difference matter?
I LIKE the Stratagene kit, works every time, but it's just too expensive.
I'm going to have to try the do-it-yourself method.



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