Incomplete shearing with Bioruptor?

[Gingeneticist]

Hi all!

Shearing gDNA for NGS sample prep. I'm trying to shear my DNA into 500bp fragments using the bioruptor and have two problems:

Most importantly:
Input = 5ug of DNA in 100ul 1xTE in a LoBind 1.5ml tube
= 50ng/ul on nanodrop
Output = 50ng/ul on nanodrop, but only ~25ng/ul on bioanalyser! Does this mean only half of my DNA is sheared/detectable on DNA 1000 chip? How can I make it shear more of my sample?

Secondly, I'm having trouble getting a tight size range - the settings on the website (6x 90s OFF - 15s ON High Intensity) gave me fragments of around 1500bp. After some fiddling I got an average of 534bp with 10mins 30s OFF 30s ON High Intensity. Size range is still quite broad though, any tips on narrowing it?

HUGE appreciation for any help

[dalib]

Dear Gingeneticist,

there could be multiple factors that are affecting your results and several modification to your protocol that will improve your results.

For example, the plastic type of your tubes is not ideal for shearing in the Bioruptor. At Diagenode we also recommend using smaller tube formats for gDNA fragmentation to obtain best results.

Nanodrop and Bioanalyzer tend to give different concentration readings, so your experience is not unusual.

If you can contact me directly at [email protected], we can discuss further about your experiment and ways to optimise your shearing.

Daniel