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
High degree of technical variation
False negatives and false positives
Contamination PCR inhibitors Calibration control
A word on dMIQE
Useful References
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