Hello, in my lab, we just start to work with Evagreen.The same samples wich amplify the constitutive gene (HPRT) with specific probe in a 24 CP, using Evagreen this amplifies in a 38 CP...this is a problem for us, because we need a shorter CP, for make the relative expressión of other genes...Does someone have the same problem?
Dear Claudia, my suggestion to check the concentration of cDNA. If it is low, you can get higher Cp.
Best regards.
Hi Claudia,
Looks like you may have a problem with primer annealing. If you're using evagreen, I'm assuming you're using a different primer pair, and not a probe? Since evagreen is a dye like SYBR, it fluoresces when bound to dsDNA, and you therefore don't need a fluorescent probe to detect amplified DNA.
To explain the differences in Cp values, I would imagine that your primer set used with evagreen is not annealing very well. To fix this problem, you'll need to try different annealing temperatures, and possibly different mastermix. Otherwise, design a new set of primers (or 2 sets just to be safe). A Cp of 38 is very late and not trustworthy, so troubleshoot and hopefully you can bring that number down below 30. It's also worth trying a melt curve to determine the purity of your product :)
Cheers,
Scott
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