I ran a primer efficiency for a primer set that was previously validated to have an efficiency of 100% as practice because I thought I was having pipetting errors. The 10-fold serial dilutions (5 in total) were from a colleague and were previously used for primer efficiencies as well. I have ran two primer efficiencies for this set and got efficiencies of 69% and 76%. My R^2 values were 0.99 for both. The Tm for the primers is 60 degrees C. My PCR conditions were 95 C for 3min, 40 cycles of 95 C for 10s, 55C (or 60C for the 2nd run) for 10s, and 72C for 30s. A melt curve was also run and looked normal. Any suggestions on why the primer efficiency is low for a previously validated set?
- Try and keep your annealing temp at Tm-2C to Tm-5C
- I note the first time, unless I am mistaken that you annealed at 55C (primer Tm = 60C = Tm-5C) whereas the second time you appear to be annealing at the actual Tm
- The Tm by definition is the temp at which specific priming on average only occurs 50% of the time (as primers cannot anneal efficiently @ the Tm)
- Thus, this might well explain your lowered efficiency: Your reactions are specific but too stringent
Hi,
Is anything identical between the previous efficiency determination and the actual one? Is only the efficiency different or additionally the Ct values of the starting concentration? (if the same template was used)
You told us, that you used a dilution series from a colleague- I would prefer the preparation of new dilutions for each experiment but this may depend on the used material.
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