Does TE buffer impact on covaris shearing?

[sehrrot]

Dear all,

I used bioanlayzer after covaris shearing. I put TE buffer in DNA samples, as protocol recommends, but result was not good. I just wonder whether salt in TE buffer impact on fragment size.

Is there anyone who use water in DNA sample in covaris shearing instead TE buffer?

[ECO]

I shear in 10mM TrisHCl 8.5, and the manufacturer recommended protocols perform pretty well.

More often problems come from not enough volume in the micro tube (<120ul will give air bubbles and thus variability). What consumable are you using and what volume?

[sehrrot]

I did covaris shearing for NibleGen exomcapture library, which recommends a total volume of 100 ul with 1X TE buffer. I mixed app. 4 ul DNA and 96 ul TE buffer in microtube (6 x 16 mm, no. 520045).

To prevent bubble, do I need to increase a total volume? how much volume is recommended or suitable?

[Hamid]

TE and Qiagen buffer EB ( 10mm Tris HCl pH 8.5) generate the most reproducible results. Deionized and MiliQ water quality and pH vary from lab to lab, and it tends to affect fragmentation reporducibiltiy.
We have also increased the processing volume in the 6x16mm tube to 130ul in order to prevent the formation of an airgap during processing which can widen the size distribution of fragments.
What settings were you using, and what did the bioanalyzer trace look like?

Thank you

Hamid

[Gillow]

Covaris for fragmentation of DNA

Does any one know how much the covaris is?

[sehrrot]

Thank you all

I found what problem was. Basically, my problem was that a graph of LCL samples in BioA appeared to begin from little bit upper of the bottom unlike blood samples. It was not a fault of TE buffer. This was due to remaining small proteins during shearing.

But I found that shearing in water gave more constricted shape than in TE. I used Pfizer medical DW water rather than the other DW. It also worked very well in further steps.

Also I found that 130 ul was the best volume (it gave narrower peak) but it was too big for a tube and little bit of samples bumped out of a tube, which may contaminate a water-tank. So I chose 120 ul or 125 ul. Also 100 ul volume, which is recommended by NimbleGen exmoe capture manual, showed a kind of birdnesting in higher concentration samples (300 ng/ul or 500ng/ul)

[shurjo]

Does any one know how much the covaris is?

Approx. $45000, for the S2.