random priming in agarose, MORE

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Tue Jun 29 11:17:24 EST 1993


In article <1993Jun25.144850.8494 at mbcf.stjude.org>, bugg at mbcf.stjude.org writes:
> In article <9306170055.AA06199 at cscgpo.anu.edu.au>, Wafa.El-Adhami at anu.edu.au writes:
 
> I don't use a "kit" for this purpose.  I make up my own solutions and buy my
> own Klenow enzyme and follow the protocol suggested by Feinberg and Vogelstein,
> including their "followup" paper in which they note that one does not have to
> remove the DNA from the agarose slice to get good labelling.
> One thing that you might want to try is a "cold chase" in which, after your
> labelling step, you add the nucleotide which was the labelled one in the
> reaction (in our case, dATP, but some of these kits use dCTP) in unlabelled
> form at the same concentration as all of the other dNTPs in the labelling
> reaction.  This will allow the Klenow to make "full-length" strands which start
> at random points on your template and extend to one end or the other.  I
> usually let the chase go for 1 hour at room temp, just because it is convenient
> for me.  This may also make it easier for you to separate probe from
> unincorporated counts.
>  
> Barbara Bugg
> Molecular Pharmacology
> St. Jude Children's Research Hospital
> Memphis, TN  USA

This is a followup to my followup!  I have been asked by a couple of people via 
direct e-mail to give them the reference to the "followup" paper by Feinberg
and Vogelstein.  I have been trying to reply to Eleanor Gallo-Hendrikx, but my
mail messages keep being returned, even though I am using the "reply" command,
in which the address is coming directly from her mail message!
Anyway, I have already posted the reference to the addendum paper.  I would
also just like to mention again that ligations work very well when performed in
low-melt agarose by the method of Crouse et al. Methods in Enzymology 101:78-83
(1983).  FMC recommends this method and describes it pretty well, although I
like the original paper better.  I do not see any reason to purify DNA from
agarose gels.  However, I do use Gelase sometimes when I am planning to cut out
a band from a gel, restriction digest it with another enzyme and run it on
another gel. 
That's my two cents!
Babs
>> 



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