PCR Primer Design: Tips for Accurate DNA Amplification

In this video, I look at a key step in a PCR (Polymerase Chain Reaction) that you must address before entering the lab: primer design.

The success of your PCR heavily depends on your correctly designed primers, as a poorly designed primer can lead to wasted time, reagents, and failed experiments.

In the video, I look at the three key factors in primer design: the melting temperature, the potential for disruptive structures like hairpin loops and primer-dimer pairs, and primer specificity.

I also cover the importance of understanding the relationship between annealing and melting temperatures and how to calculate the Tm (melting temperature) using both simple and the more accurate nearest neighbour method. Additionally, I discuss how to check for unwanted structures in the primer and ensure specificity using tools like BLAST.

At the end of the video, I provide tips on enhancing PCR efficiency and specificity, including using nested primers for challenging amplifications and adding restriction sites to primers.

By the end of this video, you'll have a comprehensive understanding of how to design effective primers, troubleshoot common issues, and optimise your PCR experiments for successful DNA amplification.

Additional Reading
This video was produced with help from the following resources:
📗 - The Biosciences Glossary - https://play.google.com/store/books/d...
📗 - Molecular Biology of the Cell (Alberts) - https://amzn.to/3zDyhQu (affiliate link)
📗 - Molecular Cell Biology (Lodish) - https://amzn.to/3zyIJZH (affiliate link)
📗 - Biochemistry (Stryer) - https://amzn.to/3XJ7gTR (affiliate link)

A help sheet defining key terms is also available in the description - https://cell.drnickmorris.com/pcr_pri...

Cell Biology Play List -    • Cell Biology (Revision)  

Links:
Biosciences Glossary - https://play.google.com/store/books/d...
Help Sheet with Key Terms - https://cell.drnickmorris.com/pcr_pri...

If you found this video helpful, please like, subscribe, and share it with your classmates.

If you would like to say thanks for the fact sheet and the video, then please feel free to Buy me a coffee - https://www.buymeacoffee.com/drnickm

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