Long PCR primers

Rafael Maldonado rafael at howard.genetics.utah.edu
Mon Oct 21 13:42:37 EST 1996


On Mon, 14 Oct 1996, Andrew J. Doherty wrote:

> HI every one
> 
> I've got a PCR primer which is 76 nucleotides long, containing the c-myc
> epitope tag and three overlapping restriction sites but only 12
> nucleotides at the 3' end are complementary to the sequence I'm trying
> to amplify. Is this a feasible primer to use? 

Sure it is. I have been using oligos from 40 to 120 bp for PCR for years. 
But you will need some tricks, because the efficiency is lower than using 
shorter oligos. 

I always gel purify oligos bigger than 30 bp. (check Maniatis). 
As you mention, you may start the PCR with 10 cycles at low 
annealing temperature; after that, 20 cycles to higher temperature. 
Choose a couple of degrees below the melting temperature (calculated with 
any method) of the sequence homologous to the template for the first cycles.
Add 5-10 degrees in the following cycles.

Also, you may raise the Mg++ concentration in the PCR buffer. It is so 
easy and it gives amazing results. I have found, too, that increasing the 
concentration of the big oligos by 5X, the efficiency is also better.

E-mail me for more specific questions (take address from the signature).

Rafa

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