Covaris parameters to obtain ~200bp fragments?

[ssg1]

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?

[Hamid]

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

[ssg1]

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.

[Hamid]

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