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Pooling over-cycled libraries containing bubble products with under-cycled

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  • Pooling over-cycled libraries containing bubble products with under-cycled

    Can someone please kindly give expertise on how I should go about pooling my samples. I have done library preparation for 80 samples INDIVIDUALLY (yes I realize now why that was a bad idea). Using TapeStation, I observe some samples to have low concentrations (1 - 2 ng), and some samples to have high concentrations (up to 50 ng) that apparently were over-cycled and now have PCR bubble peaks. Clearly TapeStation will not be an accurate way for me to assess molarity to perform pooling.

    1. Should I clean up the 10 samples that have significant bubble products? I am reluctant on this as in doing so would introduce bias as not all samples will now be treated equally prior to pooling.

    2. How would you go about pooling the samples into 1 library? Doing qPCR to quantify all 80 samples would be painful, so I am curious if anyone has ideas or experience with this.

    Note, this library prep is specific for microRNAs and my target peaks are high 150s bp and the bubble products above 300bp.
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