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I know that one of Covaris' claims to fame is that their AFA shearing is DNA concentration independent. They have literature showing bioanalyzer traces of identical size distributions obtained from 50ng and 2ug samples. Has anyone tried to shear more than 2ug of DNA in a single treatment? Any thoughts about whether the size distribution will be altered if I try to shear 10ug+ in a single treatment with parameters that were optimized on 1ug samples?
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I have sheared from 1-5000 ng with similar results. Covaris recommendation is for 100-5000 ng. Trial with your DNA is the best way. I would suggest normalising sheared DNA to the same concentration for all samples (if using various concentrations) before Bioanalyser analysis. -
Thanks for the info, nucacidhunter. I did a trial with non-precious gDNA and I got equal shearing using samples containing 1ug and 5ug of DNA (as quantified by picogreen). However, samples containing 10ug and 20ug of DNA ended up with a broad, almost bimodal distribution of fragment sizes.
In the future I'll simply break up my high-mass samples into 5ug chunks and shear each chunk independently. I bead-purify after shearing anyway, so it will be easy to re-concentrate the total fragmented pool.Comment
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Covaris shearing of DNA is indeed concentration independent. I have attached one customer's data in which they carried out a shearing of 1-50ug of DNA in 130ul volume in a microTUBE.
As Nucacidhunter suggested, I would also advise that you determine a working range of concentration for your samples, and try to keep your samples within that range so as not to bias or adversely effect the efficiency of other steps in your library preparation.
Thank you
hamidComment
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Also, keep in mind that Covaris has different tubes for different applications. I haven't looked into it specifically for your wants, but you might find a different tube type more accommodating for your needs.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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