DNA labelling kits

bhjelle at carina.unm.edu bhjelle at carina.unm.edu
Fri Sep 4 15:30:56 EST 1992


In article <1992Sep4.115359.1633 at gnv.ifas.ufl.edu> afc at gnv.ifas.ufl.edu writes:
>A few years ago we switched from labelling by nick translation to
>labelling by random priming.  We use BRL kits for both.  Besides 
>being easier to use and more reliable, random priming gives about
>100x higher specific activity.   Mostly we are probing total cell
>DNA (from single mosquitoes) for mitochondrial or ribosomal genes, 
>so probe should be in excess.
>
>My question is: why don't we see a 100x increase in hybridization
>intensity?  RP probes give a little better results than NT probes,
>maybe 2x or 3x in our hands.  
>
>I am guessing that probe length might be a problem: NT probes have a
>several hundred bases of unlabelled DNA with the labelled DNA
>attached.  RP probes have six unlabelled bases attached to the label.
>Depending on how long the synthesized strands are, this could make a
>big difference.  If so, would playing with primer concentration help?
>What about other labelling kits?
>
I had a lot of problems with random hexamer priming. Exactly
as you describe. You get unbelievable specific activity.
In my case, probing mammalian DNA, some genes would produce
no bands or only very faint bands despite the great specific
activity. In my case, this result was much worse than I
had obtained with the more humble nick-translated probes.

I always assumed it was due to the multiple priming events that
were a very short distance apart. When I ran the probe on
a gel, it was composed of very small fragments. This might
be fixable by lowering the stringency of hybridization and
washing,  or by priming with, say, 10mers, but I just moved
on to RNA probes which worked better in my hands than any
DNA probe.

If someone tells you that they are getting good results with
a particular labeling protocol, always do the following:

1. Ask them what kind of organism they are probing. What works
for phage, E. coli, or yeast may not work well for insects.

2. If the genome size of their organism is >= yours, get their
recipe.

Brian



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