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cfDNA fragmentation to short molecules
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 http://genome.cshlp.org/content/earl....full.pdf+html
and can't obtain same results as in publication on gDNA too (SMASH2 method).
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 http://genome.cshlp.org/content/earl....full.pdf+html
and can't obtain same results as in publication on gDNA too (SMASH2 method).