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The Wolbachia Project
Discover the Microbes Within

Lab 3: Polymerase Chain Reaction

Goal:
To screen for Wolbachia symbiont DNA in the extracted DNA from arthropods using one of the most widely used biotechnology techniques in biological research: Polymerase Chain Reaction (PCR).

Learning Objectives:
Students will amplify DNA extracted from experimental and control arthropods using PCR. Upon completion of this activity, students will use and understand the steps of PCR, utilize DNA as a diagnostic tool to discover microbes and barcode organisms, and seamlessly transition their discovery-based science from organisms to molecules.

Prerequisite Skills: Prior practice with micropipettors.

Teaching Time: 45 minutes

Recommended Project Guide

This lab activity requires two separate PCR reactions (Arthropod and Wolbachia) which allows for customization of the annealing temperature. The protocol is recommended for more sensitive Wolbachia detection and downstream analyses, such as Sanger sequencing.

 

Project Guide: Standard PCR (10/21) (.docx / .pdf)

 

 

Alternative Protocols

Duplex PCR

This lab activity condenses the PCR protocol into one duplex reaction that amplifies both arthropod and Wolbachia DNA in the same assay. It will amplify most, but not all, Wolbachia as the annealing temperature is optimized for the arthropod DNA. The protocol is recommended for efficient use of classroom time and resources.

  • Student Guide: Duplex PCR (6/21) (.docx / .pdf)

Quick Tools: PCR Calculators

PCR Calculators can be used as a Quick Guide at the bench. Simply download the calculator, select the preferred PCR reaction (tab), and adjust the # reactions in Column C to calculate the Master Mix volume.

  • PCR Calculator: PCR template using Promega GoTaq Green Master Mix (10/21)
  • PCR Calculator: PCR template using MiniOne Taq Master Mix (10/21)

FAQs

Q: Why do the protocols have different annealing temperatures?

A:The arthropod COI and Wolbachia 16S primers have slightly different annealing temperatures. The arthropod band will amplify best at 49°C whereas the Wolbachia band will amplify best at 55°C. Therefore, we recommend two separate PCR reactions for optimal results. However, it is possible to set up a duplex reaction (both primers in the same PCR reaction) if time and resources are a concern.

  • The duplex reaction has been tested with Promega GoTaq (M7122), MiniOne (M6208), Fisher (K1081), and NEB (M0484S) – all Taq polymerases will often, but not always, amplify both bands using an annealing temperature of 49°C. If you use a higher annealing temperature (55°C), the CO1 band may not be visible.
  • PCR reactions using GE Illustra PureTaq PCR beads work best with a 55°C annealing temperature for both primers.
  • You may run this lab with a different Taq polymerase. If you use a different reagent, please test it beforehand to make sure you get the expected results. If you don’t get expected results, try using a different annealing temperature, between 45°C and 55°C. The denaturation and extension temperatures should remain the same.