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How to avoid secondary structure in primer - (Jun/10/2005 )

Hi All,
i want to do simple 1-base site directed mutagenesis for my gene cloned in pET28 with C-terminal His-tag, cloned in NcoI and XhoI sites, i want to mutate stop codon before XhoI site, problem is i couldn't get a single primer without strong secondary structure even if i e-design it through various softwares , and my primer with sec str didn't produce anything in PCR, i've checked all other parameters, nothing seems wrong with them, somebody got any clues what might be happening.


Hi nbj,

Site directed mutagenesis can be a real pain like that with no alternatives other than using primers with secondary structure. Two suggestions that may help
1/ Use of additives in your PCR reaction such as Betaine (most companies call it something special such as Q solution or GC enhancer or something) but just get it from Sigma cheap. This can help reduce secondary structure in your reaction, other additives may also help.

2/ The second suggestion is instead of doing a site directed deletion design forward/reverse primers either side of the base you want to delete and PCR the whole plasmid including insert (using a good proof reader), phosphorylate and blunt end ligate the PCR product (vector). By doing this way you can shorten your primer and therefore reduce the secondary structure.

Couple things to try, hope they help



Have you tried changing additional 1 or 2 bases which help to reduce secondary structure of your primers (check with the software)? Remember these two additional bases changed should not resulted residue conversion or you will get an unwanted mutation!

Hope this helps