How would I verify whether custom adapters have annealed properly? I get odd results when trying to quantify using QuBit, PicoGreen or TapeStation. Since the majority of the adapter is still single stranded, is there even a way to tell?
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As you have done, Qubit or PicoGreen is the easiest and very informative. With increasing annealing efficiency you should see increasing value in concentration of dsDNA. I regularly use this method as a QC for adapters with full length or partial length complimentary regions to ensure batch to batch consistency. Obviously, adapters with short complimentary region will have lower florescence, but it is sensitive enough to discriminate between annealed and non-annealed oligos. One also can calculate theoretical expected concentration for partially or completely complimentary oligos for any given annealing efficiency.
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Originally posted by nucacidhunter View PostAs you have done, Qubit or PicoGreen is the easiest and very informative. With increasing annealing efficiency you should see increasing value in concentration of dsDNA. I regularly use this method as a QC for adapters with full length or partial length complimentary regions to ensure batch to batch consistency. Obviously, adapters with short complimentary region will have lower florescence, but it is sensitive enough to discriminate between annealed and non-annealed oligos. One also can calculate theoretical expected concentration for partially or completely complimentary oligos for any given annealing efficiency.
Originally posted by jwfoley View PostYou could also try denaturing them to see how much that reduces their fluorescence with a dsDNA-specific dye.
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What kind of values have you seen with say a full TruSeq adapter?
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