Below is a picture of a 1% gel of my qPCR products from salt marsh sediments (I know, its not the best picture). Standard and NTC are also labeled. I am wondering if my bands are too blurry to be considered a successful qPCR? The optimal annealing temperature for my primers is 53C (E = 92% - 93% consistently) and primer concentrations are set at 0.3uM. Samples contain 2ng of DNA. If I increase the annealing temp, the efficiency drops considerably. My annealing and extension are set at 1 minute and this seems to work best as opposed to 30 seconds. Melt curves have one prominent peak in both my standards and samples (A few samples have slight shoulders to the prominent peak but nothing that stands out as a clear second peak). The NTC forms primer dimers much later in the cycles as seen on the gel but don't appear in the standards or samples. So, can I accept these results or should I optimize more?
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