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  • Covaris parameters to obtain ~200bp fragments?

    I am using the Covaris to shear my samples during sample prep. My target fragment size is ~200bp, however practice shears assessed on the bioanalyser have shown smears which are very spread out, with fragment sizes of 150-250bp making up only 30% of the total fragmented DNA. The parameters used are:

    Duty Cycle: 10%
    Intensity: 5
    Cycles/Burst: 200
    Time:180

    We have also tested variations of these parameters: 10%dc, 5i, 200cpb, 120s ; 20%dc, 5i, 200cpb, 90s ; 20%dc, 5i, 200cpb, 120s and all produce very similar results. Could anyone tell me the best parameters to use in order to get a greater peak at around 200bp with a narrower spread?

  • #2
    Dear ssg1,

    The settings of 10%dc/5i/200cpb/180 seconds is the correct settings for generating 200bp fragments. The same setting is also used and cited in the Agilent SureSelect protocol.
    Please check the following:

    1. Make sure that you are using 1-5ug/120ul of DNA in the Covaris MicroTube with the AFA fiber.
    2. The shearing should be carried out with the water tank temperature between 4-8 degrees Celsius.
    3. please check to make certain that the water level in the tank during processing is as suggested in the shearing protocol http://www.covarisinc.com/pdf/pn_400056.pdf
    4. please analyze an aliquot of the sheared DNA prior to any purification or concentration step.


    If possible can you send or post a gel image of bioanalyzer trace.

    Thank you

    hamid

    Comment


    • #3
      Dear Hamid,

      Thanks for the response, we are already using 3ug/100ul, with a tank temperature between 4-8 degrees Celsius and the appropriate water level in the tank. I have attatched an image of the bioanalyser trace below.
      Attached Files

      Comment


      • #4
        Dear ssg1,

        The wider distribution that you are noticing is an aritifact of the bioanalyzer electropharogram, and it is due to the following:
        1. Please check your DNA concentration. Typically when we run an 1-2ul aliquot of sheared DNA ( 3ug/120ul)on the Bioanalyzer, we obtain FU units of around 100-150.
        2. Please increased your sample load volume on the Bioanalyzer. Based on what you are currently seeing, I would suggest doubling or tripling the sheared DNA volume you are loading on the chip.
        3. Alternatively, you can always run an aliqote on a 2% agarose TBE gel, and post stain with EthBr.

        Thank you

        Hamid

        Comment

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