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Table 3 TPCR conditions for cloning both gRNA into pCFD4d plasmid. The denaturation-annealing-elongation cycle was repeated 30 times

From: Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach

Temperature (°C) duration  
98 30 s Initial denaturation
98 10 s Denaturation
61 1 min Annealing
72 7 min Elongation
72 7 min Final extension
10 Storage