Polymerase Chain Reaction, better known simply as PCR, has come a long way in the past 30 years.
For those of you old enough to remember the not so ‘good old days’ when PCR was manually performed using a series of water baths and overlaying oil onto our precious samples (yes, seriously!), I think most of us do know how lucky we are…
The thing is though; I think we’ve begun to take PCR for granted. We perform PCR day in day out and it’s a dependable part of our lives; actually we could do one in our sleep, right? The problem with this is that one day it will turn around and bite you in the backside when you’re least expecting it.
What I mean to say is that by going back to basics we can avoid many of these potentially painful mishaps ever occurring. Taking these small and rudimentary steps will allow us to develop a much better, and quite possibly more meaningful, relationship with PCR.
And what better to help you with this task, than a copy of The Bitesize Bio Guide to PCR, a comprehensive but bite-size guide to your PCR experiment.
This e-book covers many of the fundamental rules we should all adopt in standard PCR runs and delves into more complex considerations that are essential for qPCR and digital PCR (dPCR) users.
See the below excerpt for a example of what this book includes
Extract from book: Troubleshooting an Unknown PCR product
Amplification of unknown, or non-specific products, is a common problem in PCR, and is virtually always due to lack of primer specificity. It is extremely worthwhile to determine the potential non-specific binding of the primers to the background genomic template or to other mRNA species, before purchasing the primer set. You can do this quite simply in silico using the NCBI Basic Local Alignment Search Tool (BLAST) tool.
Even if you do check primer specificity ahead of ordering, sometimes spurious bands will still appear on the gel (hence the necessity for in-lab optimization!). This could be due to the primers binding to undefined sequences or some type of contamination
of your sample. There’s good news though: chances are you can still “rescue” your PCR by adjusting some of the PCR program parameters:
• Increase the Ta; this increases the stringency of primer binding, so they should only anneal to exact matches in the template.
• Decrease the primer concentration so there are fewer opportunities for these sequences to bind to the incorrect target (this will also reduce the chance of primer dimer formation).
• Reduce the extension period, thus decreasing the time the DNA polymerase has to extend non-specific products (if they are larger than the desired product).
• Reduce the amount of DNA template added to the PCR reaction to achieve a cleaner product. Too much DNA can literally ‘get in the way’ of the polymerase, impeding its access to the target sequence.
• Reduce the number of PCR cycles – it is possible that the non-specific products are amplified after the desired amplicon. This tactic can be particularly effective in combination with reducing the amount of DNA template added to the reaction.
If none of the above work, using alternative PCR methods such as nested PCR or touchdown PCR may be more successful.
How to use this e-book
You can use this PCR guide to trouble-shoot where your gel electrophoresis went wrong or why your PCR product didn’t amplify. Use it as the lab handbook for treating your precious samples with care, methodical PCR set-up and (of course) effective primer design.
The book includes a handy PCR troubleshooting table (and link to a printable version) that you can keep on hand to help fix your PCR problems.
What’s more, if you are new to dPCR, check out the handy tips and advice on performing PCR using the recently developed droplet dPCR platform. This is a fantastic resource for new-users and will guide you through specific steps for maintaining droplet count and dPCR assay optimization.
Finally, remember to take care of your PCR experiment and it will likely take care of you!
You can pick up your own copy of the e-book, The Bitesize Bio Guide to PCR, here.