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[yaeldeb]

I am a relatively new qPCR user.  I started with an applied biosystems machine and I did about 5 standard curves with a bunch of primers till I realized that oops, my dillutions were off!  Then I got things working well, and I got standard curves with good efficiencies -3.3--> -3.6.  Then our department got 2 new BioRad machines that are across the hall.  I thought I would switch over.  I'm using a fast mix from applied biosystems.  I tried a standard curve with the same primers and the efficiencies were much lower.  I thought maybe it was my cDNA quality - so I used a kit for RNA and got great ratios with nanodrop for RNA.  The efficiencies were a bit better - but still above 3.6.  I spoke to the rep from BioRad and he suggested doing a gradient for annealing temp for the primers to find the best temp for this mix on their machine - I feel like that will waste a lot of reagent and time.  Does anyone have any tips??  If not I might just go back to the old machine (even though its two floors down and you have to sign up in advance).
Thanks!