need your help!

Mr. M.J. Lush mlush at hgmp.mrc.ac.uk
Fri Nov 27 04:55:19 EST 1998


In article <73l8na$74b$1 at news.iastate.edu>, Min Du <dumin at iastate.edu> wrote:
>Hi, 
>I am doing experiment in DNA labeling, because I just begin to do, I am 
>not sure wnat I am doing is correct.
>I have generated a 425 fragment by PCR and want to label with Digancin by 
>ramdon primer method. Is it OK?

        425bp may be a bit short to label by random priming...
on average a random primer will produce a probe half the lenth of
the template.

	I'm not familier with 'Digancin'  but I assume that it is some sort
of labeled nucleotide which is incorporated into DNA via a DNA polymerase
(since it is a random priming method).
 
        Since your labelling a PCR product,  why use _random_ primers?
You could use one or both of you PCR primers to prime the incorporation 
reaction,  then your probe DNAs should all be 425bp long.

	You could go one stage further and use a linear PCR.
ie add the Digancin nucleotide to a PCR reaction mix which has
all the normal PCR reaction componants but only one of the PCR
primers cycle 10 times and you should end up with lots of single
stranded labled probe (Make sure you use the PCR primer complementory
to the RNA though,  or you could get a rarther low singnal on your
Norhtern :-}


>Last time, I generate a probe with this method and failed to detect any 
>signal in Northern blotting. Can you give me any suggestion?



-- 

Michael
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
NPC rights activist           |      Nameless Abominations are people too.



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