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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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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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