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Lab 4: Gibson assembly of HCAII into pETblue2 vector and transformation of E. coli with the reactions

Lab 4 Objectives

  • Prepare DNA for Gibson Assembly
  • Use a Gibson Assembly reaction to clone the HCAII gene into the pETblue2 expression vector
  • Transform E. coli with the cloned DNA using the heat shock method
Fig. 4.1. Outline of the Gibson assembly protocol. Two pieces of DNA have matching sequence (shown in red). Within a Gibson assembly reaction, these DNA pieces undergo several steps: Exonuclease chews back 5′ ends, DNA fragments anneal, DNA polymerase extends 3′ ends, and finally DNA ligase seals the nicks.

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Biochemistry 551 (Online Version) Lab Manual Copyright © by Lynne PROST is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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