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Old 01-04-2011, 06:47 AM   #1
lzembek
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Location: Cambridge, MA

Join Date: Dec 2009
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Default Shearing Amplicons with Covaris

Does anyone have experience shearing 3-4kb amplicons down to ~500-800bp size range (suitable for 454 LC) with the Covaris? I have the E210 series instrument, and would be using the 100uL microTUBE crimp-cap. Any settings or data you have would be really useful, thanks!
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Old 01-05-2011, 08:34 AM   #2
Hamid
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Location: San Diego, CA

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Default

Hi,

In the document attached to this email, you will find methods for shearing DNA to different targeted fragment sizes using micro-tubes with or without crimp caps.

You have an option to either use the protocol for 400bp or the one for 500bp (I believe the 400bp protocol will serve you better):

1) For the 500bp fragment target, the method recommends 80 seconds - but I will recommend to you that you perform a time course where you use 80, 90, 100 and 120 sec to better optimize your process since you will be using PCR product for starting material.

2) For the 400bp fragment target, the method recommends 55 seconds - but I will recommend to you that you perform a time course where you use 55, 65, 75 and 90 sec to better optimize your process since you will be using PCR product for starting material.


Also, please feel free to check our website for the latest protocols available. http://www.covarisinc.com/supported-protocols.html

Just to follow good lab practices, may I remind you of the following:
1) To get a distribution where the target fragment has by far the most predominant percentage, please use 130 μL (not 100 μL) in the micro-tube. Filling the micro-tube with 130 μL of sample will help avoid the presence of any air gaps which may create two phases where one phase will contain completely processed sample and the other phase will contain sample that was not processed to completion.
2) Use TE or 10 mM Tris-HCl at pH 8.0 for best and reproducible results.

Please let me know if you have other questions or need more suggestions.
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