re-amp of PCR -- problems

[Rafael Maldonado]

On 11 Jan 1995 zool201 at csc.canterbury.ac.nz wrote:

> To whom it may concern
> 
> I'm trying to reamplify a very low concentrartion of primary PCR
> product and am ending up with smears every time.
> I don't want ot re-purify the primary product because of the very low 
> concentration.
> 
> Any hints would be greatfully accepted.
> 
> Andrew Holyoake 
> PhD student
> University of Canterbury
> Christchurch 
> New Zealand

Andrew, several things can try to improve your PCR
-Use different concentrations of Mg++ in the reaction (this applies for 
the first reaction too). Choose the reaction with less smearing. 
-Rid of the template of the first PCR, and other unwanted secondary 
products: gel purification, size fractioning, etc. 
-Use other internal primers to amplify in the second reaction. 

I hope this helps

--------------------------
Rafael Maldonado
University of Utah
Rafael at genetics.med.utah.edu