mutations in primer sequence - (Jun/19/2008 )

Hi,

Most of the time when I find mutations after ligation/cloning or QuikChange, they are in or near the primer sequences. What is probably causing this? Bad primers? Bad annealing? I typically use oligos from 22 up to 60 residues, only desalted.


Thanks!

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did u checked the conditions of ur primer may be one of ur primer make hairpin or loap and that coud make mutation in ur pcr product

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Bad luck is the answer if they are in the primer sequence. Primer synthesis isn't perfect. Most primers are fine but some primers are mutant and usually missing a base pair. You just got unlucky that the single PCR product you cloned into your clone happened to be made with one of the few mutant primers. Isolate another clone and 99% of the time the new clone will be fine.

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