I have a problem with my set-up primers. my results are very poor and I don't know what is the problem for example if results of patients for the specific virus is positive with a viral load of 115000 when I test it with my designed primer probe it shows a 100 copy. I changed the primer concentration, and the volume of the qPCR master mix and also I used other master mixes with better-quality enzymes but the problem was not solved. what do you think I need to test to enhance the results?
Dear Haniye Salimi
Please check the efficiency of your primer by a standard curve method. Every time you receive a new set of primers, especially when using SYBR Green chemistry during quantitative polymerase chain reaction (qPCR), you should always run a standard curve to calculate the efficiency of your PCR primers. This should be helpful
https://barricklab.org/twiki/bin/view/Lab/PrimerEfficiencyqPCR#:~:text=You%20want%20to%20achieve%20primer,really%20throws%20off%20this%20calculation.
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