I am working with CD4+ cells isolated from 15-20 year old HIV + PBMCs in DMSO stored in LN2 freezers with minimal freezing and thawing. I'm running a digital droplet PCR on the extracted CD4+ DNA and analyzing the amount of intact and defective proviruses in the samples and get high DNA shearing results.

Protocol:

The shearing index (measured by the RPP30 gene ) is consistently high (> 70%). I've limited the number of freezing/thawing events after extraction, minimized vortex steps after lysis, and added carrier RNA, all with no effect. I've mostly been experimenting with various conditions in the genomic extraction protocol as I think the DNA would be relatively protected from shearing forces while inside the cell.

Ideally, the shearing would be around 30% or less. Has anyone else had these problems or have ideas to minimize DNA shearing?