End-labelling problem

ALPE Pagliuca pagliuca at cds.unina.it
Tue Jun 2 18:58:39 EST 1998


Hi everybody. This was just to submit to you a problem that i encountered
when i tried to get a radiolabeled probe for emsa assays.
I began by cloning a double-stranded 30-mer into pBluescript and then
cleaving on flanking sites and finally filling in the 5' protruding end
with Klenow and alpha32PdCTP. When i resolved the labelling reaction on a
native PAGE, it seemed that all the radioactivity was incorporated on the
vector and none on the insert. Please note that i checked that the
digestion was complete. After repeating the experiment a couple of times to
get really sure that there were no simple mistakes, i decided to make a
different test: I just cut the plasmid with one enzyme flanking the insert,
then inactivate the Klenow and precipitate, then cut again with two enzymes
whose
recognition sites are on opposite sides on the polylinker with respect to
the site that was used to generate  the radiolabeled ends, then resolve on
a PAGE to see whether the two ends were labeled with different efficiency.
Though the result may seem paradoxycal, with some enzymes i observed a
different efficiency in some cases; hope this will be useful to some.
Ciao
Alfredo



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