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May 22nd: I've just received some very interesting feedback from a colleague, who found that switching to a two-step protocol of 72° and 98° for the secondary PCR reaction dramatically increases the efficiency of his projects. In other words, ignore the calculated annealing temperature and go straight to the extension step. If anyone out there has been struggling with super low efficiency, please give that a try and
shoot me an email
to let me know if it works better for you. If I get some more positive feedback, I'll make changes to the website.
-Steve
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