Random primed labelling trouble

Eric Lader lader at ambion.com
Thu Sep 26 22:44:58 EST 1996


In article <jnakamot-2609960144290001 at news.ucla.edu> Jon Nakamoto,
jnakamot at ucla.edu writes:
>
>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). 
>

The template is a bit small for RP, but I have labelled fragments down to
150 bp without a problem.  Also, random decamers worked better for us and
are also in our RP kit. One thing that helps random priming in general is
snap freezing after boiling the template with the primers.We recommend
either powdered dry ice or liquid nitrogen. Ethanol baths sometimes get
into the tubes when you quick chill them.  This may make a big
difference. Also, if you are using 4degree label, limit the amount in the
reaction to 5 ul. We quite often see inhibition of klenow with 10ul of 4
degree label (ie: redivue) For best results, we use the -20 stuff.

How are you assessing whether the reaction worked? Better to do TCA.
Maybe the fragments you get when you lable a small template are getting
hung up in your spin column. We can routinely electroelute from agarose
and dialyze away the TBE and label with no problems.



               **my opinions are just that**
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        Eric Lader Ph.D.	Senior R&D Scientist,  
                          Ambion Inc.
Unique tools for Molecular Biology Research

Phone (800) 888-8804       Fax (512) 445-7139
				  (512) 445-6979       lader at ambion.com
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