Schumer lab: Primer efficiency testing

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  • See primer design section for primer design. PCR fragments should be between 70-100bp. We usually design four sets of primers per gene of interest.
  • Before testing primer efficiency you should use a regular PCR kit as instructed to make sure the primers amplify bands at the expected length.
  • Check that peaks are clean and around the right size with the tapestation. See D1000 TapeStation Protocol.docx in the Protocols folder in Box or email for the protocol and all necessary reagents and consumables.
  • test efficiency of all primers that amplified as expected! It can be tricky to get a gene of interest and house-keeping pair that are efficient at the same annealing temperature. Or a gene of interest and a single-copy gene pair!
  • Make sure it's okay to use the qPCR machine in the Fraser lab
  • To test efficiency you need to run triplicates of each primer on at least 6 serial dilutions of cDNA or gDNA. If the DNA is too concentrated you will need to dilute it. 10ng/ul seems to be fine but less is also fine!
  • the recipe for one qPCR reaction is 5ul of iQ sybr green supermix + 3.4ul of water + 0.3ul of 10uM forward primer + 0.3ul of 10uM reverse primer + 1ul of DNA
  • efa1 a swordtail housekeeping primer we have in the lab is efficient at 62C so I usually start there for annealing temperature. Use the cycle CFX_2stepAmp+meltcurve.prcl.
  • Check in the biorad software that there is a single peak in the melt curve analysis for each primer. If there is more than one peak per primer, then the primer cannot be used at the this temperature.
  • Use this template to calculate efficiency /Box/Schumer_lab_resources/Extractions_and_library_preps/qPCR data/efficiency_calculation_template. Make sure the standard deviation between triplicates is low. Exclude outliers from analysis.
  • Efficiency should be between 0.9-1.10. If your efficiency is too high, increase your annealing temperature. If your efficiency is too low, decrease your annealing temperature.
  • Once you have a primer pair that are in an acceptable range of efficiency you can proceed to your samples of interest