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

In article <9502221351.aa23246 at>, betts at ORION.ETSU.EDU

> 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
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  But, this paper is a little out of date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                  Queen-Shear Heart Attack

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