AMOUNT OF PRIMER IN DNA FINGERPRINTING

Richard E. Showalter show at agouron.com
Tue Feb 28 20:12:20 EST 1995


In article <9502221351.aa23246 at etsuodt.etsu.edu>, betts at ORION.ETSU.EDU
(GORDON BETTS) wrote:

> I am about to order some primer for an undergraduate fingerprinting exercise
> I am trying to duplicate and need to know how much primer to use.  I will
> use the D1S80 primer.  The procedure by Budowle et al. (Am. J. Hum. Genet.
> 48:137) calls for 1 uM.  Isn't this a lot?  When I look at order forms, they
> often have a checkbox for 50 or 100 nM.  Lot of difference!  
> Thanks!
> Gordon Betts
> Biology Dept.
> East Texas State University
> Commerce, TX 75429-3011
> 903/886-5369
> ============================================================================
> ======
> Life is like a bowl of stew, you need to stir things up once in a while so 
> the scum doesn't rise to the top.
>       Author unknown
> ============================================================================
> ======

   First of all this is not fingerprinting, DNA or otherwise.  This
protocol is refered to as Amplified Fragment Length Polymorphism (Amp-FLP)
analysis.  Sorry, I have been reading too much of the alt.fan.oj
commentary.  I was responsible for setting up the D1S80 system for the
Calif. DOJ DNA crime lab, although I am no longer in Forensics, I still
keep up on the subject and would be glad to assist you if I can.  The
Budowle paper is correct in that 1 micromole of each primer is used per
PCR reacton.  This is about what you would use for DNA sequencing so I do
not think that it is too much.  But, this paper is a little out of date
now.  They used 100ng of Genomic Human DNA for the amplifications.  This
is 10x more DNA than is necessary for a proper amplification, and can
result in amplification artifacts, remember the monomorphic band mentioned
in the paper.  5-10ng is more than enough DNA to amplify and should be
quantitated accurately.
   I have found that the amount of primer purchased from most vendors in
the 0.5-1.0 OD unit range to be more primer than I can use in a life time
for most experiments.  You generally get far more actual OD units than the
amount you are paying for.  In fact I use a company called Genosys located
in your state for all of my oligos.  
   If you have any more questions please feel free to E-mail me, I have
protocols and tips that may help save you some grief.

-- 
Richard E. Showalter             "Hey Hey Hey Hey it was
Senior Associate Scientist        the DNA.  Hey Hey Hey Hey
Agouron Pharmaceuticals, Inc.     that made me this way."
show at agouron.com                  Queen-Shear Heart Attack



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