Biotin Pcr primers and anealing temperature

Duke Groebe drg at prophet.pharm.pitt.edu
Fri Aug 23 07:55:22 EST 1996


In article <4vi08d$blo at pulp.ucs.ualberta.ca>, karl at hobbes.chem.ualberta.ca
(Karl Voss) wrote:
> 
> Hi,
> 
> Does anyone know if having 5' biotin on pcr primers lowers the anealing 
> temperature that they need.  I have been getting very good pcr results with
> a non-biotinilated primer set that have short 5' sequence overhangs to be
> used for directional cloning.  I wanted to directly sequence the pcrs so 
> I got a biotinilated primer made that did not have the extra 5' bases.
> The resulting pcr bands are very weak.  I have quantified the primers 
> carefully and assume that they are of high purity (they were purchased from 
> Research Genetics).
> 
> Has anyone run into similar situations?  If so I would like to hear
> from you.
> 
> Thanks to everybody for reading this.


I had a primer made with 5' biotin attached.  I assayed for the optimal
concentration of MgCl2 and found that virtually no rxn occurred at 1 or 2
mM MgCl2 but got very good results around 9 mM MgCl2.  You might try higher
concentrations of MgCl2, especially if you suspect that the Tm is lowered
by the presence of biotin (raising the concentration of the primer will
also stabilize interaction with the template).

Good luck

Duke Groebe

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