covaris protocol for large input mass gDNA?

[amplicus prime]

Hi everyone,

I've been working on shearing gDNA down to 180-200bp to use as a control in sequencing experiments. I've used the standard protocol to great success with 5-10ug input mass, but now I would like to be able to use more than 10ug of starting material - perhaps 50ug up to even 100ug per microtube (130uL) so I can make large quantities of the sheared gDNA at one time. Covaris FAQ states that if you plan on shearing more than 30ug, you may need to increase treatment time and increase temperature to 20C, where I suppose the external chiller unit would not really be needed. Since these darn microtubes are so expensive, and with limited gDNA I'm working with at the moment, I was hoping somebody has already done this and has some recommended treatment times for the aforementioned input mass (between 30-100ug)? I'll assume all other shearing parameters will not change from the standard <1.5kb shearing protocol for the given target size.

Cheers,

A.P.

[JackieM]

Did you ever find a solution to this? I'm looking to fragment some larger quantities as well. It'd be really nice -cost wise- if we were able to do it in a single tube.

Best,
Jackie

[Hamid]

Hi Amplicous,

Too much DNA in a small volume of 130ul will render the viscosity too high and a controlling variable. You can shear >50ug of DNA in 2ml volume using the Covaris milliTUBEs.

Thank you
Hamid