fragmentation of ovine DNA

[Laruo]

Hi,

this is the first time I will construct a DNA library to next-generation sequencing. I'm working with ovine DNA and trying to set up the fragmentation step in a Bioruptor Pico machine. I need a library with insert size of 350pb. I have tested several different conditions and 30"ON/30"OFF and 16 cycles is the best one. However, I have observed differences between replicates of the same sample, and also between different samples. In addition, I would need to get a closer curve in the Bioanalyzer in order to loose the least amount of DNA after the cleaning step. I have tried increasing the number of cycles to 18, but it doesn't work.

Can anyone suggest any idea? have you also observed this lack of reproducibility?

Thanks!

[kwaraska]

The lack of reproducibility has more to do with the BioRuptor itself than with the DNA type.

We switched to a Covaris because of this very issue- it was not reproducible and very difficult to get exactly what you want.