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Old 05-21-2015, 07:48 PM   #4
Ingeneious
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Location: Los Angeles, CA

Join Date: Dec 2014
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Quote:
Originally Posted by nucacidhunter View Post
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.
What kind of values have you seen with say a full TruSeq adapter?

Quote:
Originally Posted by jwfoley View Post
You could also try denaturing them to see how much that reduces their fluorescence with a dsDNA-specific dye.
That's a good idea. I tried something similar by comparing the annealed(?) product versus an equimolar mixture of unannealed oligos. With a Qubit dsDNA BR kit it did not seem to make a difference - they all seemed to generate a similar value. With PicoGreen I believe I received roughly double the concentration. However, even the individual ssDNA oligos generated a value.
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