Standard Curve

[CPPnerds]

I'm trying to generate a standard curve for actin gene expression in candida albicans. I'm doing a 5X dilution curve. This is my 6th attempt but i still fail to get parallel curves for my graph. I did everything i was supposed to. I even vortexed and span down the templates of every single dilution. So far my curve is a mess and my NTC on the other hand has been constantly producing fluorescence. Is there anybody who had encountered the same problem as myself before? Is there anything i can do to get better results?

[amberpdidit]

Are you diluting the RNA? Maybe your quality isn't what you think. I would check RNA quality (not just quantity, if you have access to a bioanalyzer that'd be good)

The other thing I would consider is doing smaller dilutions, maybe 2 fold. You may just be in a wierd range for detection.

Good luck, this stuff can be so frustrating.

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Wait, I just saw that your NTC is coming up. That is a big problem. I think you have some contaminant somewhere. Start with your water and other buffers and if you are seeing something in NTC than you need to fix that.

Are you doing 1-step or 2-step? TaqMan or SYBR?