Lab 3: Analysis of PCR Products and Preparation for Gibson Assembly

Lab 3 Objectives

  • Determine whether the PCRs you set up in Lab 2 resulted in DNA fragments of the correct size
  • Prepare for next week’s Gibson assembly reaction to clone the HCAII gene into the expression vector pETblue2 by:
    • digesting PCR products with DpnI to remove template DNA
    • purifying the digested DNA
Fig. 3.1. 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 Lab Manual Copyright © by Lynne Prost. All Rights Reserved.