User:Mbennie/Notebook/Papers and Books/Methods

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Error fetching PMID 15659713:
  1. Error fetching PMID 15659713: [PCR-mutagenesis-restenzymes]
  • Uses type II restriction enzymes to rapidly induce desired mutations into DNA
  • Designs two primers around mutation site with type II (Ear1, BsmA1, BsmB1, Sap1, Ksp6321, BpuA1, Alw261, Eam11041) restriction enzyme sites
  • Performs PCR to create two strands of DNA with mutation and restriction enzyme sites (100ng of template, 1 uM primer pair, 200 uM dNTP, 2.5 U of Taq polymerase)
  • Cuts DNA with restriction enzymes and ligates back together (10 U enzyme, 400 U T4 DNA ligase)
  • Performs gel purification to get desired product
  • Cuts and ligates into a plasmid
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