random priming -->> increased probe size??

Andre Hamel hamel at ccu.umanitoba.ca
Sat Jun 19 11:21:49 EST 1993


In article <9306191424.AA01000 at deimos.med.pitt.edu> bsh at MED.PITT.EDU (Basavaraju Shankarappa) writes:
>> 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
>
>	If the size of the probe generated is a problem, may be you can try
>one other alternative.  Since processivity of enzymes is one of the 
>criteria determining the size of the probe, why not try the most processive
>enzyme known (sequenase??) in the random primer labeling.  If anyone is
>already using this system, can we learn of your experience?
>Raj Shankarappa
>bsh at med.pitt.edu
>


Klenow is pretty "crappy" compared to T7 DNA pol for processivity ... thus
random prime labelling is better (get more incorporation in side by side
labelling comparison .... awhile back we realised this when our Klenow ran
out, had plenty of Sequenase II on hand ... to no surprise it worked and
so far consistently appears to be superior to Klenow ... mind you Klenow
does tend to "die off" more readilly than most NON-thermo stable DNA pols
(for example T4, TdT and T7/Sequenase). ... couple of occassions that we
ran such Sequenase random primed probes on mini gel -> autorad ... appears
as though there's more "fuller length" probe, but then again, there was
noticeably more incorporation overall (usually > 30% more than the couple
of Boehringer Klenow "dregs" remaining in those vials used in those
particular comparisons) ... autorads of those couple of mini gel checks
of such probes appeared as inverted "tear drop", with uppermost portion of
signal being almost full length, about 1 kb ... reminder that these
were "denaturing" agarose mini gels (1% formaldehyde in gel and tank
buffer ... sample heated in 50% formamide 65C/10 min, 50 ng/uL final conc
EtBr added to sample, heated again 10 min/65C, dye mix added, loaded ...
same done for Hind III Lambda size marker ... so estimated sizes
presumably reflected ssDNA.

regards, Andre

                            ********************
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                     FAX: (204) 945-8062     
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
                            ********************



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