Fragmentation Methods

[kbushley]

Wondering if anyone has any informed opinions/experiences to share on which fragmentation method (nebulization, sonication, NEB fragmentase) gives the best results for fragmenting whole genomes (~ 35MB).

[basickler]

We used nebulization for a while and it worked pretty good, though you turn half your input DNA into an aerosol. But if you have access to one, I'd use a covaris sonicator. No DNA loss, and the band is tight enough that you can only use one gel instead of two.

[shurjo]

I use Covaris for RNA and have been very satisfied with it. Other folks in our group also use it routinely for genomic DNA and have almost stopped using the nebulizers altogether.

[runemoe]

We have used the Bioruptor (sonicator). It works well, but we need a lot input material (5µg). We are considering buying a Covaris,
as this makes it possible to sonicate at a narrower range, hence lowering input.

[greigite]

I have used both covaris and nebulization, and of those two, covaris is definitely the winner: much less messy, no sample loss, tighter control of size distribution, plus the ability to get much smaller fragments than possible with nebulization. It also worked well with very small sample quantities (nanogram level). Haven't tried the fragmentase yet. Main drawback of covaris is the very high cost (instrument + $5/tube).

[MACHR]

RNA fragmention using Covaris

Hi Shurjo,

I have been told that RNA can't be fragmented by sonication. I use a Covaris for DNA shearing and would love to be able to fragment my RNA using it too. Can you advise on protocols/general methods you use?

Thanks!

[shurjo]

My preferred settings are as follows:

For 150ng of polyA RNA in 120ul of DEPC-treated water in a 6x16mm Covaris tube:

(10% duty cycle; 5 intensity; 200 cycles per burst; 60 seconds) gives a peak centred approximately around 200bp

If you want smaller or larger peaks, the time in seconds is the variable to play with.

HTH,

Shurjo