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Old 11-02-2017, 09:41 AM   #1
Junior Member
Location: Russia

Join Date: Feb 2013
Posts: 8
Exclamation 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
and can't obtain same results as in publication on gDNA too (SMASH2 method).
Attached Files
File Type: pdf Ion shear.pdf (123.3 KB, 5 views)
File Type: pdf NEB_fragm_titr_po_MgCl2_2017-11-01.pdf (2.06 MB, 4 views)
File Type: pdf custom buffer_gragm_2017-11-02.pdf (2.16 MB, 1 views)
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