PCR failures can be frustrating but understanding the reasons behind them is crucial for troubleshooting in your workflow or application. It's important to approach troubleshooting systematically and avoid compounding issues "on the fly" by varying only one component at a time.
This approach can save you significant time, effort, and resources, potentially even millions of dollars and pounds. Based on our experience of manufacturing PCR reagents over the years, systematic PCR troubleshooting often reveals that the issue lies within these areas 99% of the time.
While it may be easy to blame the PCR reagent first, several billion commercial PCRs have taught us that it's more likely to be related to:
1. The NAP method
2. Poorly designed assay or suboptimal cycling conditions, including empirically determined reagent enhancements such as increased Mg2+, DMSO, etc, in conjunction with adjusted cycling conditions.
3. PCR instrument issues, usually related to ROX, fluorophores (for real-time PCR), or age-related performance failure of Peltier if using an old instrument for conventional PCR.
PCR always requires experimental validation, and that's the beauty of it when approached in a controlled manner.
By taking a systematic approach and referring to independent reviews, such as the deep dive review from Labome, you can develop a deep empirically determined understanding of your personal PCR workflows, which is where AMPED wants you to be.
Remember, troubleshooting PCR can be complex, but with a systematic approach and the right resources, you can overcome challenges and optimise your PCR protocols for successful results in your research.
LABOME METHOD - PCR PROTOCOL & TROUBLESHOOTING