Random primed labelling trouble

Ned Mantei mantei at neuro.biol.ethz.ch
Fri Sep 27 06:13:49 EST 1996


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. 

220 bp should work. You might want to look at Suganuma and Gupta, Anal.
Biochem, 224:605, "An evaluation of primer length on random-primed DNA
synthesis for nucleic acid hybridization: longer is not better". They
conclude that random hexanucleotides are better than longer primers. 
Incidentally, our recipe is 1 ng DNA fragment and 1 uCi of one
32P-nucleotide per ul of reaction volume, and 0.25--1 ng labelled fragment
per ml of hybridization solution. Don't bother removing unincorporated
nucleotides after the reaction--they don't interfere.

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



More information about the Methods mailing list