random priming in agarose

[Martin Kennedy]

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

> 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


Wafa-

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!


Cheers,

Martin

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