DNA Shearing Results Covaris M220

[erlandsen]

Hi,

I have just started shearing my DNA for downstream exome library preparation.

I used the illumina recommended protocol on the Covaris M220 instrument, aiming at peak top at ~250 bp.

Then I checked the samples on High Sensitivity chip(bioanalyzer) and got the results as in the attachment.

Of what I have seen in other threads it looks generally ok, but I puzzled about the shift in the "graph shoulder" on some samples?

Any comment on the results in general and on the issue I pointed out will be appreciated.

Ragards,
see

[addyblanch]

I think they look correct, the movement in the "shoulder" would just be down to the amount of fragments at that size. They'll never all be the same but as long as the average size is your target, it'll be fine.

[Anna Maria]

DNA shearing results in M220

Hi,

It looks that you overloaded the HS Bioanalyzer chip hence the "funny" shape of the peaks. When performing the analysis of DNA fragments on the High Sensitivity chip it is very important to use the small amounts of DNA to prevent overloading of the chip that causes all kinds of migration artifacts.
When the proper amount of DNA is loaded the top of the peak should be below the top of Lower and Upper Markers. In your case the sample peak is dwarfing the markers. I am quite sure that if you dilute your samples 1:10 and run them again all peaks will look the same.
Your DNA shearing was good, the problem is the analysis of fragmented DNA.

Anna

[cement_head]

Hi,

It looks that you overloaded the HS Bioanalyzer chip hence the "funny" shape of the peaks. When performing the analysis of DNA fragments on the High Sensitivity chip it is very important to use the small amounts of DNA to prevent overloading of the chip that causes all kinds of migration artifacts.
When the proper amount of DNA is loaded the top of the peak should be below the top of Lower and Upper Markers. In your case the sample peak is dwarfing the markers. I am quite sure that if you dilute your samples 1:10 and run them again all peaks will look the same.
Your DNA shearing was good, the problem is the analysis of fragmented DNA.

Anna

Yep, that's what we've found - don't overload the HS DNA chip.