Lab 2: PCR Amplification of HCAII and pETblue2

Lab 2 Objectives

  • Amplify the HCAII gene and the pETblue2 vector via PCR using primers appropriate for Gibson Assembly
  • Determine whether the amplification reactions resulted in DNA products of the expected size

In Lab 2 we will simulate using the polymerase chain reaction (PCR) to amplify the HCAII gene (which encodes the enzyme Human Carbonic Anhydrase II) and the pETblue2 vector, and running the PCR products on an agarose gel. In lab, you will start by working with a partner to design primers for both PCRs. You will then work together to complete a table summarizing each step in the procedure and its purpose. You will then complete virtual lab situations and watch a video of the lab procedures. You will finish by again working with your partner to determine the sizes of the DNA bands you expect to see on the gel. In a future lab, we will simulate using the amplification products in a Gibson assembly reaction in order to clone HCAII into pETblue2, an expression vector suitable for protein production in E. coli.


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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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