Radio-labelling EthBr stained DNA

[bugg at mbcf.stjude.org]

In article <9202160236.AA03507 at genbank.bio.net>, mhollowa at LIBSERV1.IC.SUNYSB.EDU (Michael Holloway) writes: 
>         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

No, the Feinberg and Vogelstein method does not use sheared ssDNA.  They used a
set of random hexamers from P-L Biochem (#2166).  We used the same thing for a
while and now we use Pharmacia's #27-2166-01, which is still the same thing!
There was an addendum to the original paper, which came out in Analyt. Biochem.
137 (pp 266 - 267) in 1984 in which they pointed out that the DNA did not need 
to be removed from the gel.  This addendum has some good "recipes" for the 
buffers used in the protocol.  
Also, I would like to point out that, in our lab, NuSieve works just as well as
SeaPlaque.  You do need to remember that it is usually used at 2-4%, while
NuSieve is usually used at 1%.  Thus, you need to add more water in order to
dilute the agarose.  I have had good results with all of FMC's pure low-melt
agaroses.  Good luck!                   
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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
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