random priming in agarose

Wafa.El-Adhami at anu.edu.au Wafa.El-Adhami at anu.edu.au
Thu Jun 17 05:55:03 EST 1993


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




More information about the Methods mailing list