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Old 11-03-2011, 04:49 PM   #1
wanderingneuroscientist
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Default Covaris S2 shearing for small amounts of DNA

Hi all,

I was wondering if anyone has a protocol for generating 200-300bp fragments from small amounts of DNA (50-70ng) using Covaris. It took us awhile to get something working for 500ng and most of the standard protocols offered seem to be for >1ug.

Cheers!
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Old 11-04-2011, 05:06 AM   #2
henry.wood
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We've used the standard settings for small (<50ng) amounts of DNA and made 200bp libraries for the illumina without too much problem. My understanding is that the covaris doesn't over-shear if you give it less DNA. You need to reduce the amount of adaptor later on.
They were just the settings that we got given when we bought it a couple of years ago. Reading the rather faded (and smudged - so might not be accurate) sticker on the side of the machine, they are:
25 cycles of-
Duty cycle 19.9%, intensity 9.9, 1000 cycles per burst
Duty cycle 15%, intensity 8, 500 cycles per burst.
Other people might have a more up to date protocol, but this works for us.
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Old 11-04-2011, 09:14 AM   #3
Hamid
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Hi,

Please use the same settings as for using 1-5ug of DNA in 130ul. The technology is quite concentration independent below 10ug of DNA when processed in 130ul in our microtubes.
You can find the settings located at http://covarisinc.com/pdf/pn_400056.pdf
The settings provided by Henry is unfortunately unnecessarily too high. You will risk breaking the microTUBES using his settings.

Thank you

Hamid
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Old 11-04-2011, 10:32 PM   #4
wanderingneuroscientist
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Thank you Henry and Hamid. Hamid - my labmate tried different concentrations (100ng - 3ug in 130ul) with the same protocol and obtained different shearing patterns, but I'll give it a shot and see what happens in my hands. Henry, do you have any advice on how the concentration of the adaptors should be adjusted? Thanks again.
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Old 11-05-2011, 08:18 AM   #5
Hamid
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Hi Wandering....,

Please note that DNA has to be sheared in the Covaris glass microTUBEs. Shearing in plastic will not work reproducibly, or efficiently as acoustic energy is absorbed by the polymers and transferred as heat to your sample.
If you take a look at pages 5-7 of the shearing protocol I forwarded to you in my last post, you will not that concentration does not affect shearing efficiency, average fragment size or distribution. That has been tested exhaustively at countless sites.

Thank you

Hamid
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Old 11-07-2011, 01:24 AM   #6
henry.wood
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Hmm, best ignore my covaris advice it seems. As for adaptors, I don't know what protocol or indeed machine you are working with. Just from an illumina point of view, since that's what we use - if your protocol is designed for 1ug DNA and you only use 50ng, then you will have a vast excess of adaptors and get a nasty adaptor peak in your library. If you proportionally reduce it with the DNA concentration then it should be better.
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