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NGS PCR Cycle Quantitation Protocol

NGS PCR Cycle Quantitation Protocol

7 Comments
  1. Hi Ethan. Above link reports a “404 – file not found” error. I would be very grateful if you were able to re-post

    Many thanks,
    Aaron.

  2. ethanomics permalink

    Hi Aaron,
    Thanks for the heads up. I must of deleted the file on accident. The new link should work.

    Ethan

  3. Sweet, thanks Ethan. Our NGS lab is currently navel-gazing over lib quantification, and struggling somewhat with over-clustering, so any helpful tips – like your qPCR protocol, for example – are very much appreciated. BTW, what machine do you run the KAPA fast mix on?

    Aaron.

  4. ethanomics permalink

    Aaron, great idea for a new post. This is my opinion on the current status of library quantitation: https://ethanomics.wordpress.com/2012/02/08/library-quatitation-science-or-voodoo/

  5. Hi Ethan. I’m hazy on the “determine the number of cycles it took to get to 50% of amplification” part of this protocol, and would be very grateful if you could please provide a little more detail.

    Many thanks,
    Aaron.

    • ethanomics permalink

      That’s because it isn’t really the best description. Let’s see if this makes more sense. Looking at the amplification curve in the linear scale (i.e. non-log scale) with the fluorescence on the Y-axis and cycles on the X-axis. I take the Ct when the fluorescence is 50% of the maximum value, which should be about 1 cycle before you see the slope start to flatten out.

      Hope that makes more sense.

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