Protocol Cold Spring Harbor Laboratory Press on January 25, 2018 -Published by http://cshprotocols.cshlp.org/ Downloaded from www.cshprotocols.org ii. Stage 2: Eight cycles of denaturation for 40 sec at 94°C, annealing for 30 sec at 57°C with an increment of 1°C/cycle, and elongation for 1 min, 45 sec at 68°C with an increment of 1 sec/cycle. www.cshprotocols.org 3 CSH Protocols Cold Spring Harbor Laboratory Press on January 25, 2018 -Published by http://cshprotocols.cshlp.org/ Downloaded from

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... ww.cshprotocols.org 4 CSH Protocols iii. Stage 3: 22 cycles of denaturation for 40 sec at 94°C, annealing for 30 sec at 65°C, and elongation for 1 min, 30 sec at 68°C with an increment of 1 sec/cycle, followed by a final extension for 3 min, 40 sec at 68°C.