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
As researchers, we are constantly on the lookout for new and improved ways to analyze, detect and quantify our favorite protein or gene. Luckily, we don’t always need to reinvent the wheel! PCR-ELISA is a good example of where two commonly used techniques have been merged together to create a very powerful analytical tool. What…
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In a recent article, I gave some tips about how to obtain good results with sequencing DNA after bisulfite conversion (it contains some tips that apply to the approach described in this article, too). Bisulfite sequencing is a very useful technique if you want to know the methylation status of every CpG in your genomic…
DNA shuffling uses PCR technology in a very creative way. It allows you modify your protein to make a new protein you want. You can evolve proteins in microcentrifuge tubes on your very own lab bench. Isn’t that fantastic? DNA shuffling is also a very powerful technique for directed molecular evolution. W. Stemmer first used…
Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss…
10 Things Every Molecular Biologist Should Know
The eBook with top tips from our Researcher community.
