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For site directed mutagenesis, plasmid template was diluted 100-fold, mixed with 0.04 U/L Phusion DNA polymerase (Thermo), 0.2 mM dNTP's, 0.5 M forward and reverse mutagenesis primers and 1x Phusion reaction buffer (Thermo), and then placed in a thermocycler for 98C for 30 s followed by 20 cycles of 10 s at 98C, 20 s at 70C, 4.5 min at 72C with a final extension at 72C for 10 min

It degrades according to the actual conditions it experiences

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