Some kits like Quick change II demands HPLC or PAGE purified primers over desalted one. Can any one explain the effect purification and does it depends on primer length as NEB recommends Purification of oligos more than 55 bases long.
It doesn't really matter much. I have used non purified oligos (59 bps) for side directed mutagenesis and it worked perfectly fine for me.
Hi there,
Yes, HPLC or PAGE purification consists in considering only the final product exhibiting the right size. So this step mainly avoids the side effect of the presence of unwanted primer in your reactions. So for mutagenesis experiments it's better to use the purified version in order to improve the yield of the mutagenesis.
Primer purification used for the mutagenesis depends on the kit supplier.For example in case of gene art site directed mutagenesis( Invitrogen) the desalting of the primers is sufficient for mutagenesis.Primer length should be around 45 bases.
At PrimerGenesis, we use desalted primers for most site-directed mutagenesis applications. If using long primers (90nt or longer), PAGE purification is recommended.
For automated primer design, check our software at: http://primergenesis.com
Happy mutagenesis :)
Bruno
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