random priming -->> increased probe size??

Basavaraju Shankarappa bsh at MED.PITT.EDU
Sat Jun 19 12:19:19 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?

	As usual, Andre has us all confused!!!
If I can venture a guess from the following, I think he is saying that
incorporation is better along with longer probe lenghts, using sequenase.
I guess for certain purposes, where kinetics of hybridization are important,
may be it is better to obtain more of smaller sized probes as opposed to
few molecules of longer probes.
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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