Understand and Troubleshoot PCR with The BitesizeBio Guide to PCR

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