illumina library forget USER
I am working on a library using NEB adaptor and I forgot use the USER enzyme. could it be still work?
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I doubt that it would work properly. Since the NEB adapters are hairpins opened by USER, the ligated fragments will probably re-anneal preferentially over primer binding, since they're covalent circles. Once priming and extension happen, you could get exponential amplification, but it would likely be low complexity and biased because of the previous effect. -
Well. I got a nice DNA yield after amplification and purification. Using the amplified products, I performed another round amplification for 30 cycles by taking a few ul as template. And I got three major bands at 100, 200 and 300 bp, together with a smear whole lane staining. No obvious staining above 1.5 kb. Just using Ethdium bromide staining.
I believe it won't as good. And this just a test for what it might turn out without USER. I wonder what is the major bands showing up after great amplification.Comment
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