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Top 9 reasons why your assay isn’t working

By: Roche Life Science

Posted: | Lab Life - Real-Time PCR


Here are nine reasons why your assay might not be working correctly.

  1. Check the machine. Are the time and temperature parameters set correctly on the thermocycler? This is the equivalent of IT telling you to reboot your computer. It’s easy to identify if a parameter is set incorrectly, allowing you to move on quickly.
  2. Sequence and structures. Sometimes the primary and secondary structures can be the cause of your troubles. If possible, choose an alternate region further upstream or downstream. If you’re limited to a specific region, try using longer primers or additives that reduce the melting temperature of the DNA.
  3. Wrong reagents. Are you using the correct reagent for your assay? If so, you could be experiencing reagent degradation, which can happen to dNTPs and Taq polymerase.
  4. Primer concentration. Do you have the correct primer concentration for the assay?
  5. Pipette calibration. Pipetting accuracy is critical to the success of your experiment. The easiest way to check your accuracy is by weighing water. Water has a known density of 1 g/mL, so each microliter (mL) should weigh 1/000th of that, or 0.001 g. If you have a 100 mL pipette, you should have 0.1 g of water.
  6. Operator error. It happens, as much as we don’t like to admit it. If you’ve struggled with getting your assay to work, ask another researcher to run a sample with your template and primer to compare to your results.
  7. Suboptimal conditions. Recommended annealing temperatures aren’t always ideal. Try reducing the temperature to below the primer melting temperature, to diminish nonspecific products, or raise the temperature to exceed the production of nonspecific products. Better still, use a gradient cycler and test a range of temperatures in 1°C increments.
  8. Sample quality. While the best DNA sample is a fresh sample, we know that’s not always possible. At the very least, look for a sample that has undergone a minimum number of freeze/thaw cycles. To test the quality of your sample, perform a series of standard dilutions to your DNA. Running PCR on your diluted samples will give you the limits of your reaction. Low values indicate contamination, which will require you to run a cleanup protocol with a readily available cleanup kit
  9. Too few cycles. Insufficient amplification can be the result of too few PCR cycles. A good starting point is 20-35 cycles, though fewer cycles can be used when template concentration is high


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