I am trying to find out good standard for hIL22 using sybr green technique, but everything I tried gave bad slope, bad efficiency and multiple melting curve. Its hard to find good primers for hIL22. My lab follow sybr green technique and I shall stay with established technique but its troublesome for me. Can anyone suggest any solution? Can I do taqman pcr? How do I publish my data when i have taqman for one gene and sybr green for rest of the genes?
multiple melting curves indicate contaminants and non-specific amplification. it is also possible that the annealing temperature is set too low leading to the mispriming of primers to unwanted gene segments. I suggest you redesign the primers or, look for journal articles with established primer sequences for your gene.
May I ask if you are following the two step or three step pcr? also, i suggest you read on standard curve relative quantification method which corrects differences in efficiencies of both the endogenous and target gene.
http://blog.mcbryan.co.uk/2013/06/qpcr-normalisation.html .
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