Random primed labelling trouble

Jon Nakamoto jnakamot at ucla.edu
Thu Sep 26 03:34:25 EST 1996


Oh, I'd love to hear the definitive answer on this one (somebody, please!)

Isn't 220 bp a bit short to be random-primed labeled using decamers? I
think that hexamers work better, but I confess that we've had variable
results even with these. Your idea of rapid re-annealing has some appeal,
and I wonder if some of the tricks which help in direct sequencing of PCR
products might help here, too (e.g., snap cooling after boiling of
primer-template mixtures, driving reactions with high primer
concentrations, etc). 

I'd like to hear how most people label their short fragments...



In article <52ambt$ln6 at merkurius.lu.se>, Fredrik Kamme
<fkhero at biogen.wblab.lu.se> wrote:

> Hi everybody,
> I've got a random primed labelling trouble:
> I'm using a DNA labelling kit with random decamers (MBI-Fermentas) that
> works fine on Qiaquick gel eluted fragments. However, when I try
> labelling a 220bp DNA piece (also gel eluted plasmid insert) I get no
> labelling. Tried it twice, but no success. Is the DNA piece too short and
> re-anneals too quick? Can there be a contamination in the gel-eluted DNA?
> 
> Many thanks in advance, Freddy

-- 
Jon Nakamoto
jnakamot at ucla.edu



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