Lab 2: PCR Amplification of HCAII and pETblue2
Lab 2 Objectives
- Learn how to design primers for Gibson cloning
- Lab safety and check-in
- Practice with pipettes
- PCR to amplify the HCAII insert and the pETblue2 vector
This lab will begin with a chance to use online databases to find vector and insert sequences, followed by a primer design exercise. Although you will use the primers provided by the instructors for the PCR, this exercise will help you to understand how to design primers.
After the primer design exercise, you will be introduced to working in the wet lab by reviewing safety protocols, doing an equipment check-in, and practicing using pipettes to accurately measure liquid volumes.
Next, we will use the polymerase chain reaction (PCR) to amplify the HCAII gene (which encodes the enzyme Human Carbonic Anhydrase II) and the pETblue2 vector. In order to set up these reactions, you will need to design primers with appropriate sequence overlap for Gibson assembly. In lab 4, we will use 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.