random priming in agarose

Martin Kennedy mkennedy at chmeds.ac.nz
Sat Jun 19 00:20:40 EST 1993

In article <9306170055.AA06199 at cscgpo.anu.edu.au>, Wafa.El-Adhami at anu.edu.au 

> 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


The size of the probe you get relates to the frequency with which the random
primers anneal to the template DNA.  The kit you are using probably suggests
adding around 30ng of DNA to the reaction;  the concentration of random primer
in the reaction should then be such that on average priming occurs every 200bp
or so.  If you use small (less than a few hundred bp) template DNA fagments,
then your probe size will be smaller on average.  The same holds if you add
less than the suggested optimum amount of DNA into the labelling reaction
(because you effectively increase the primer to DNA ratio).  If  you are 
adding the recommended amount of a 700bp fragment, and everything seems
to be working, then don't worry!



NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
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