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Mutagenesis and primer purity? - (Oct/31/2007 )


I am going to do a site-directed mutagenesis using QuikChange method(whole plasmid PCR). On strategene manual, it says primers should be at least purified by PAGE or HPLC. Has anyone had any problem with using primers that are just purified by standard desalting? Thanks a lot!!


We have always gone for primers purified by standard desalting. It works even for mutagenesis.

Good Luck !!!


I mostly go for gel or HPLC purfied as I ended up having mutants that lacked some bases due to incorporation of a "shorter" (or not complete primer). The extra cost of the purification for me was less than the cost of having to miniprep and sequence more clones. (I couldn't include a Restriction site to check the primers being complete or not).


In my experience, it pays to get the HPLC or PAGE-purified oligos as it saves a lot of (potential) resequencing. With standard de-salted oligos, the best case is that it works while the worst case is that you'll have to reorder (and wait for) purified primers. Save the time and order HPLC or PAGE-purified oligos.


Thanks all!! I didn't expect the purification was so expensive... $40 for each primer...