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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