I´m trying to do a mutation of one aminoacid on a protein, I designed the primer acording to the protocol for Site-Ditected Mutagenesis and I got a false positive. On the sequencing of the obteined DNA it shows that some stop codons where added after the sequence of the primer ends. It did perform the mutation correctly and everithing look fine before and some aa after, but just when the sequence of the primer ends the DNA also changed and produced a stop codon.
the error rate in pcr is very temperature dependent so if the primers are annealing at a low temperature then the polymerase is already extending but making more errors which wil be close to the end of the primer at the annealing temperature. Try annealing at a higher temperature.
While designing primers, be careful with frameshifts. Enzyme site like NotI (which have 8bp recognition sequence) in the cloning primers may result in frame shift. Recheck your primers.
hi
you remember that sequencing efficacy between is 400-600 nucleotide , so you can do :
1- clone the pcr product in T-vector and sequencing by specific vector primers for reduce the reading error of sequencing ( for example T7-R , T7-F primers / pGEMT-easy )
2- Another way to get more precision and ease can be use from gene synthesis method .
good luck
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