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Old 06-07-2019, 07:07 AM   #1
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Location: Kansas

Join Date: May 2019
Posts: 4
Default NEB Fragmentase mitogenome advice

Hi, I am currently working with mitogenome sequencing of small mammals. I have amplified 5 PCR amplicons of the mitogenome around 4-5kb fragment lengths. My next step is to pool fragments and conduct shearing using NEB Fragmentase before library prep. Does anyone have any recommendations of the timing of incubation to achieve around 200bp fragments? My plan is to conduct a time series trial to find the optimum time, however, I am wary that low concentration of PCR Fragments that after shearing I may no actually be able to visualize on an agarose gel to confirm fragment shearing size. Conc of fragments ranges from 5ng/ul - 250 ng/ul.

Currently looking at end result using 300bp paired-end MiSeq kit for sequencing hence the 200bp fragment target.

But I am interested to know peoples experiences or recommendations to consider.

thanks for the help

Last edited by frascom; 06-07-2019 at 07:26 AM.
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fragment analysis, nebnext ultra, shearing

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