In response to the ongoing disscussion about Random Priming...
If you use 5ul of 32P-dCTP, the theoretical maximum yield of
labeled DNA (assuming 100% incorporation) is 20ng!
Therefore, if you load 100ng of cold template DNA into your
labeling reaction, you will end up adding 16% label and 84% cold competitor
into your hybe mix. The more template you add, the worse it gets, and this
is assuming 100% incorporation. If you use 20ng template, you will end up
with 50% label and 50% cold competitor. Adding more template only gives
weaker probe. My experience (with the Amersham kit, the DuPont kit, the
boerenger kit, and with home made reagents) is a drop in the efficiency
of the reaction if less than 10ng is used. That leaves an optimal
window of 10ng to 20ng DNA, and an acceptable window of 5ng to 50ng
of template DNA.
Another little note, some old work by one of the companies showed
that the reaction is more efficient with labeled 32P-dCTP. I don't know
why, but they showed reaction kinetics for all 4 labels using a variety of
As for primer length, shorter primers give less efficient priming,
but have a better chance of finding a match. I have found 6 to require
slightly longer reactions, but always give good labeling. 8-mers are
quicker, but I sometimes am not sure if I should trust them. 14-mers
gave good, fast reactions, but sometimes did not label certain probes
very well. These lengths were tested because they were supplied with
certain kits that we had tried.
I am now using a home made "
I am now using a home made "kit" and we have added a cold extention
reaction after the labeling reaction. This lengthens the hot probes,
increasing both the total amount of label and the size (hybe efficiency).
I think one ofthe commercial kits also uses this trick.
Finally, I never separate out my un-incorporated nucleotides.
When the reaction works, the efficiency is so high that it is a waste
of time and money to separate. When it doesn't work, just start over
and do it right. Separation unincorporated nucleotides from trash
will not turn that trash into gold. Every time it has not worked for
me it has been because of old Klenow. Gotta be nice fresh stuff.
Dr. Leonard N. Bloksberg
PreissJ at clvax1.cl.msu.edu
Dept. of Biochemistry
Michigan State University