Understand and Troubleshoot PCR with The BitesizeBio Guide to PCR

Written by: Kirsten Hogg

last updated: March 16, 2023

Over the years, I’ve had my fair share of ups and downs in the lab. The latter quite often centering on a failed or plainly weird PCR experiment. As I’ve gone on and become ever more fastidious about my lab practices I’ve realized that the majority of these little calamities were perfectly avoidable. In my new e-book, The Bitesize Bio Guide to PCR, I aim to impart much of my hard-earned and entrenched wisdom with readers, who like me, prefer a simple PCR life. Standard PCR revolutionized our ability to study a small fragment of DNA or RNA by replicating its number thousands-fold. This small leap was only the beginning, however, and paved the way for much bigger and better technology to come our way. Today, while many researchers and clinical services still depend on standard PCR techniques, the majority of us are also seeking highly precise and quantitative solutions, including quantitative real-time PCR (qPCR) and more recently digital PCR (dPCR). This e-book is here to help you figure out the basic practices that are essential for getting your PCR, qPCR or dPCR experiment right. By focusing on the ‘how to fix’ element, this e-book provides an essential PCR trouble-shooting guide for any student or researcher using PCR in the lab. And here’s a little sneak preview of the e-book contents:

Chapter 1 Introduction

Main types of PCR What this book will do for you

Chapter 2 Standard PCR

No bands
     Missing or non-optimized PCR reagents      Gel electrophoresis      DNA polymerase      GC-rich targets      Primers      Sample quality Weak bands      Reaction components      Non-optimal PCR program Extra bands      Primer dimer formation      Unknown PCR product      Bands appearing in negative controls  Alternative PCR methods      Nested PCR      Touchdown PCR and Stepdown PCR      Multiplex PCR General PCR troubleshooting guide Useful References

Chapter 3 Quantitative real-time PCR (qPCR)

Hydrolysis (Taqman) probes Dual Hybridization (FRET) Probes SYBR Green Dye No fluorescent signal
     qPCR assay set-up 
     Primer/probe design 
     Plate preparation Low sensitivity (high CT values)      Biological samples      Primer design      qPCR assay set-up Non-specific amplification      Primer dimer formation      Poor primer specificity      Genomic DNA contamination       High degree of technical variation            Program set-up      Plate set-up Poor standard curve            Sample handling      Program and plate set-up Useful References

Chapter 4 Digital PCR

Droplet digital PCR Low droplet (partition) count
     Blocked micro-channels      Empty wells      Droplet damage/loss
Low fluorescence amplitude/threshold      Poor assay optimization      Inappropriate fluorescence threshold      Template concentration
High degree of technical variation False negatives and false positives      Contamination      PCR inhibitors            Calibration control A word on dMIQE Useful References

Conclusion

You can pick up your own copy of the e-book, The Bitesize Bio Guide to PCR, here.
Kirsten Hogg

Kirsten gained a PhD in Reproductive Biology from the University of Edinburgh.

More 'PCR, qPCR and qRT-PCR' articles

pcr pitfalls

PCR Pitfalls: The Devil is in the Details

ByEmily Humphreys

PCR was actually one of the first lab techniques I learned as an undergrad. Despite being sometimes labeled as a pretty basic lab skill, PCR doesn’t always work as expected. This “fickle” success is due to small details or hidden hazards within the PCR workflow that can cause your seemingly uncomplicated experiment to fail.  This…