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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

Conclusion

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

 

1 Comment

  1. KK on October 22, 2014 at 4:00 pm

    Hi Kristen, I liked the Ebook content. Newbie like me would definitely get helped from the hard work you put in writing the Ebook.I would like to access the E-Book, Do you have the link where I can download. Thanks a Ton

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