random priming in agarose

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Fri Jun 25 15:48:50 EST 1993


In article <9306170055.AA06199 at cscgpo.anu.edu.au>, Wafa.El-Adhami at anu.edu.au writes:
> Hi there,
> I am using the Amersham megaprime DNA labelling system (based on the method
> developed by Feinberg & Vogelstein) for labelling DNA fragments excised
> from LM agarose gels.  It is working very well.  However, my concern is the
> size of the probes the system generates.  The system claims that the system
> will synthesize no larger than 200 bp product, but at the same time the kit
> describes that the probe length is dependent on the size of input DNA (as
> well as other factors).  My concern is the size of input DNA since I am
> making probes from fragments as large as 700 kbp and not sure if it will
> still make only 200 bp probes! I have spoken to representatives from
> Amersham and they were helpful to some extent as much as they knew about
> the method. They claimed that because the kit uses the Klenow fragment of
> Polymerase, certain concentration of primers and nucleotides(according to
> the method these should be in excess) the size should still be in the 200
> bp range. I have checked the relevant references and also did an
> electrophoresis experiment for sizing the product and I beleive I do get
> about the size they describe (200 bp), but would like to hear some helpful
> comments and views on this matter.  If anyone out there has tried this
> system or has any ideas to offer, I would greatly appreciate their advice! 
> Wafa

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
> 



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