microsatellite loci amplification

Charles Brockhouse chbrock at iastate.edu
Thu Feb 27 14:15:20 EST 1997


kfeldh1 at ICARUS.CC.UIC.EDU wrote:
> 
> I am trying to amplify lemon shark microsatellite loci using the PCR but
> haven't had much luck.  The DNA I am using was extracted from fin clips
> (stored in ethanol), by standard phenol-chloform extractions.  The
> primers being used are specific for lemon sharks (were developed from a
> lemon shark gDNA library).
> 
> We are getting plenty of DNA from the extractions, so I don't think lack
> of DNA is a problem.
> 
> I have tried many different PCR "recipes" (using DMSO, spermidine,
> varying molarity of dNTPs, varying volumes of PCR reaction, etc.) with
> no luck.  Does anyone have any suggestions?  Could something be
> co-amplifying with the DNA that is inhibiting the PCR?
> 
> Thank you in advance,
> 
> Kevin Feldheim

My experience with insect DNA is that PCR inhibition is fairly common. 
The use of phenol in the DNA extraction process often aggravates the
problem.  Two ways of dealing with it are to dilute the DNA further (put
less inhibitor into the reaction) or add BSA to the PCR reaction
(hopefully bind up any protein-binding contaminents in the DNA prep).
Good luck,

Charles L. Brockhouse
Insect Genetics, Entomology
Iowa State University
Ames, IA., 50011-3222

Tel: (515) 294-4570
Fax: (515) 294-5957



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