PCR with two consecutive mutations

[Pepa Florez Pérez]

Dear all,

 

I am trying to do a PCR using an oligo with two consecutive mutations. According to some algorithms the tm for this oligo is arount 67ºC (considering also the mutations). It was designed so there are aprox. 12 nts. on each site of the mutants that hibridize perfectly with the target sequence.

 

We have also run a PCR with hibridization temperatures of 42, 55, and 62. But it didn't work. Of couse, the positive control does.

 

Do you have any idea about why it is not working? Any further suggestion?

 

Thank you very much

 

Eva
 		 	   		  
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