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. Author manuscript; available in PMC: 2016 May 10.
Published in final edited form as: Nat Protoc. 2015 Oct 22;10(11):1820–1841. doi: 10.1038/nprot.2015.115
Cycle Description Temperature (°C) Time (s)
1 Initial denature 95 180
2 – 30 Denature 95 10
Anneal* Tm 30
Extend 72 60 per 1 kb of the amplicon
31 Final extend 72 300
32 Hold 10 infinite
*
M13F(−21) and M13R primers have a Tm of 47 °C; for other sequencing primers use the Tm calculated with Oligocalc (Box 2).