ds cDNA shearing on Covaris?

[microgirl123]

I've asked this before but I'm wondering if anyone has any new information or experience! I've gotten variable answers from Covaris tech support, different tech support people for our Apollo robot, and Illumina. As usual, the answer seems to be that we need to have tons of extra sample to test different Covaris settings on, and I just don't have that option.

Since cDNA is already shorter than genomic DNA, do the Covaris settings have to be adjusted to end up with properly sized DNA? I'm trying to end up with a peak ~220 bp.

[NextGenSeq]

No you use the same settings as for genomic DNA. We do this all the time.

[microgirl123]

Thank you! It's good to hear from "real-life" people rather than tech support. I love our Covaris, but it's really hard to adjust the settings if you don't have lots of extra sample to play with (and money for the tubes and BioAnalyzer chips to look at the shearing).

[adam.geber]

I just did several days worth of validation of the standard Covaris gDNA protocols for use with a set of cDNA fragments (1-3kb) and found that a) time is the only variable worth manipulating (i.e. simply pick a protocol and tweak the treatment time until you have an acceptable distribution of fragment sizes) and b) the final distribution is heavily dependent on the size distribution of your input sample. I ended up sonicating until my amplicons were thoroughly fragmented and followed up with an AMPure bead purification to put a consistent lower bound on my fragment sizes -- I imagine you could do the same if you're getting ready for an Illumina run.