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Standard curve issues

primer callibration machines standard curve

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#1 yaeldeb

yaeldeb

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Posted 01 March 2012 - 04:35 AM

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!





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