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Old 12-13-2010, 08:36 AM   #1
hawaii454-0
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Location: UK

Join Date: Dec 2010
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Default covaris shearing settings for paired end

Hello,
Does anyone use the Covaris to shear their samples in the Roche 3kb paired end library preparation step of circularised DNA fragmentation?

If so, what settings do you use?

I used these Covaris settings:
duty cycle - 5; intensity - 5; cycle/burst - 500; time - 120secs
and got the attached Agilent result.

Mahalo
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File Type: jpg paired end 3kb covaris.jpg (71.9 KB, 63 views)
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Old 12-13-2010, 01:38 PM   #2
Hamid
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Location: San Diego, CA

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Hi,

The settings you specified was provided in a paper by the Broad institute in which they had used PCR tubes for shearing their samples. We think that generates wider than desired distribution. Shearing in plastic PCR tubes increases the possibility of thermal induced biased shearing. It also lacks the reproducibility of processing samples in the glass microtubes.
My suggestion is that you use the setting for the 400bp fragmention stated in the protocol located at http://www.covarisinc.com/pdf/pn_400056.pdf, and carry out a time course of 10, 20, and 30 seconds beyond the specified protocol treatment time.
Since you are dealing with very low concentration samples, please analyse on a high sensitivity chip.
Please remember to process in a volume of 130ul, in TE or 10mM tris-HCl using the Covaris microtubes.

Thank you

Hamid
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Old 02-06-2012, 02:15 PM   #3
UKboston
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Before I start another thread: Does the TE or tris-HCL affect the size range or just protect the DNA?

I'm looking for ways to narrow my peaks.

Thanks
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