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Ingeneious 05-21-2015 08:06 AM

Custom Index Adapters
 
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?

nucacidhunter 05-21-2015 04:18 PM

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.

jwfoley 05-21-2015 05:22 PM

You could also try denaturing them to see how much that reduces their fluorescence with a dsDNA-specific dye.

Ingeneious 05-21-2015 06:48 PM

Quote:

Originally Posted by nucacidhunter (Post 173041)
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 (Post 173046)
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.

nucacidhunter 05-21-2015 08:24 PM

Quote:

What kind of values have you seen with say a full TruSeq adapter?
I have not done exact math. Using 10 uM (each) 40 base long adapter oligos with full complimentary bases across oligos I get around 70 ng/ul with PicoGreen and for 10 uM 30 base long oligos with 14 complimentary nucleotides 30 ng/ul.


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