Micrococcal Nuclease (Mnase) to digest ds DNA to ~160bp?

[ibuyufo]

I'm using the NEB Mnase kit (Micrococcal Nuclease, 10X Mnase Reaction Buffer, and 100x BSA along with 100 ng of genomic DNA. The final reaction volume is 50 uL and it's incubated in the thermalcycler at 37C for 5 minutes.

I've used a range from 500 to 4000 U in my test but so far I haven't been successful at generating any fragmented product. Am I not using enough starting material? Is there a protocol I can follow to generate the product that I need? Thanks for the help!

[Chipper]

Are you digesting chromatin or purified DNA?

[ibuyufo]

Are you digesting chromatin or purified DNA?

Hi Chipper,

I am trying to digest purified DNA.

[Chipper]

MNase cuts in between nucleosomes, I did not know it was possible on naked dna. Maybe try NEB fragmentase?

[ibuyufo]

MNase cuts in between nucleosomes, I did not know it was possible on naked dna. Maybe try NEB fragmentase?

Thanks Chipper. This is great and I really appreciate you taking the time to answer my question. I am not experienced doing enzymatic DNA fragmentation since a Covaris instrument was always used for this purpose. I've moved on to a new job and they don't want to spend the money to get the instrument and they've decided to go in this route.

[nucacidhunter]

KAPA hyper plus kit is also for enzymatic shearing and library prep if you are preparing Illumina libraries.

[ibuyufo]

KAPA hyper plus kit is also for enzymatic shearing and library prep if you are preparing Illumina libraries.

Thanks for the suggestion nucacidhunter! I'm actually trying to cobble up something that I can use as reference samples with different frequencies to be used in future NGS assays.