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korostin 11-02-2017 09:41 AM

cfDNA fragmentation to short molecules
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Dear All,

I'm trying to find fragmentation conditions for cfDNA shearing to obtain 35-50 bp distribution. I started with overdue Ion Shear kit, which I found in fridge and got expected results (Ion shear.pdf) on cfDNA model (mix of 100-300 bp amplicons) - 50 ng input.

Then my NEB fragmentase was delivered. After a series of experiments I have no traces in 35-60 bp range. It looks like NEB enzyme mix is too active and fragment DNA directly to ~20-35 bp pieces that are washed on column step. I tried to decrease MgCl2 concentration in reaction (attached NEB fragm.pdf - wells 1-10: 0-10 mM is MgCl2 added to reaction, 10-30-60 minutes of incubation), but there is no sufficient difference in traces. I use NEB fragmentase buffer v2. It has BSA and 15 mM MgCl2 concentration instead of no BSA and 10 mM MgCl2 in v1.
Also I tried to change buffer and prepared one without MgCl2. I fixed incubation time and added different MgCl volume to obtain final concentrations from 0 to 10 mM ("custom buffer.pdf" wells 1-4).

It looks like NEB's nuclease, which cut nicked DNA is too active. Do you have any ideas how to decrease it's activity? I'm guided by this article
and can't obtain same results as in publication on gDNA too (SMASH2 method).

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