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Hi, guys. I'm trying to construct a PE500 library from 50ng genomic DNA by using the Nextera transposase. However, the fragment size always concentrate on 200bp even I have chosen the HMW buffer. How can I control the MW of the fragments? Or is there anyone have similar experiences?
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Hi Kitty,
Not sure why you started another thread rather than continuing your old one...
From your bioanalzyer plot:
It looks like most of your library is ending up in annealed concatamers. How about denaturing it (2 minutes 95 oC, followed immediately by chilling on ice) and then running on an pico RNA bioanalyzer chip?
But if that is not possible, you could try using way less cycles of amplification but then use a DNA High Sensitivity Agilient Chip. That way you can see your library products without needing to do so many PCR cycles that your primers run low and you get high levels of amplicon daisy chaining (or bird nesting, or whatever you want to call it.)
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Phillip -
nextera for targeted exon capture?
is nextera library prep OK to do exon capture?Comment
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