Originally posted by adam.geber
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Right now I've definitely not optimized all the parameters. Library prep is definitely still a bit difficult for me to grasp.
Hmm I think it definitely could be overamplification, since it does seem on the samples that I start with lower concentration of template (just by checking gel band intensity) I tend to have less of this kind of issue. The qPCR results for these sample wasn't super high though, so it's a little hard for me to imagine the reaction being depleted of primers (it's added to the reaction at 0.5uM). Maybe I'm somehow losing amplified product to annealing due to over cycling?
I'm also using Phusion hot start for this reaction. I'm wondering maybe that has something to do with it? perhaps not enough Mg?
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