Nucleic Acid Shearing
1.
Where can I find DNA shearing protocols?
The current DNA shearing protocols are published on our web site. Click here and follow the link for a protocol for the desired fragment size.
2.
What are the DNA fragment sizes can I get using Covaris instruments?
We are currently providing protocols for the following fragment sizes: 150bp, 200bp, 300bp, 400bp,
500bp, 800bp, 1 kb, 1.5kb, 2kb, 3kb and 5kb. Typically, sizes between the defined protocols may be achieved by increasing the time of the treatment of the nearest size DNA fragment.
3.
Can I use the published protocols to shear PCR amplicons?
Yes, the PCR amplicons can be fragmented using standard protocols as long as the size difference between starting material and desired fragment size is tenfold. When the size difference is smaller the treatment time needs to be adjusted. We recommend running a time course experiment to determine the length of treatment required. As a rule of thumb, if the size of your starting material is within the size range of the generated DNA fragments you need to extend the treatment time (e.g. if you are trying to get DNA fragments with a mean size of 200bp the range of fragments that are generated is between 50 and 700bp). If your amplicon is 500bp you will need to increase treatment time from 180 sec to 200 sec or more.
4.
What happened to Intensity (as employed in previous versions of SonoLab)?
In SonoLab 7, Intensity has been replaced by Peak Incident Power to better represent acoustic treatment conditions.
In the S2 and E210 instruments, Intensity served as a dimensionless proxy for the power emitted from the transducer during each acoustic burst, with Intensity 10 corresponding to approximately 350 Watts of Peak Incident Power. New generation instruments operating under SonoLab 7 are capable of delivering up to 500 Watts of Peak Incident Power. Rather than invent a new dimensionless scale,
Covaris chose, in SonoLab 7, to specify Peak Incident Power in true Watts.
The use of Watts for Peak Incident Power (in place of Intensity) actually clarifies the setup of acoustic treatments. If 250 Watts of Peak Incident Power are employed with a 20% duty cycle, then the average, nominal power emitted from the transducer is simply 20% of 250, or 50 Watts.
5.
How do I convert a method developed on an S2 or E210 for use with SonoLab 7?
SonoLab 7 FAQ and setting conversions
6.
Can I use the microcentrifuge tube to shear the DNA?
No, the sample vial is a critical component of the acoustic circuit of the Covaris AFA process; the sample vessel material, thickness, geometry and the solution in the vessel all play an important role in

this circuit. Following the Covaris recommended protocols and using the appropriate Covaris sample vessels will provide the optimal results.
7.
The miniTUBES all look alike, can I use the clear miniTUBE to shear DNA to 3 kb fragments?
No. Although the different miniTUBES might look very similar they are in fact different and their construction is optimized for a given size of DNA fragments.
8.
Can I re-use miniTUBES?
No, to assure proper performance and avoid cross-contamination the miniTUBES should only be used once.
9.
I am not getting consistent results when shearing DNA. What might be the reason?
Inconsistent results might be caused by several different factors; however there are few important things to check first: o o o
Sample volume: In the microTUBE sample volume should be 130 µl. The presence of large headspace allows for the occasional formation of an air gap in the tubes disrupting the consistent fragmentation of DNA to the desired size range.
Water level: The water level is critical for DNA shearing. Not only does the water allow for the acoustic energy to couple from the transducer into the tube, it is also important in keeping your sample at the appropriate temperature during processing and minimizing vibrations which could lead to glass tube breakage. As a rule of thumb, the water level in the tank should be about 1 mm below the cap of the microTUBES during the processing. When using mini TUBEs the water level should be such as to cover 2/3th length of the tube.
Water bath temperature: Water temperature should be closely controlled and matched to the application. Since the warmer temperatures promote less forceful collapse of acoustic cavities within the sample fluid, causing a shift toward larger mean fragment size, the water bath temperature during the DNA shearing should be tightly controlled. When using micro TUBE and clear mini TUBE the water bath temperature should be between 6 and 8 0C. DNA shearing in blue and red mini TUBES is performed at 19-210C
Degas level of water: insufficient degas levels within the bath may result in less efficient o acoustic coupling and thereby shift the mean fragment size. System degas pumps should be run in advance and during AFA treatment. Prior to running a process the water bath should be degassed for at least 30 min for S1 and 45 min for E220.
10.
Why do I need to change water in the tank every day?
Purity of the water in the bath is very important for the proper acoustic coupling. When applying acoustic energy in rate-limited applications, foreign material such as algae and particulates may scatter the high frequency focused acoustic beam, resulting in a shift to a larger mean fragment size. Bath water should be deionized water, changed daily unless continuously treated by the optional Covaris Water
Conditioning System.
11.
Besides daily changing the water in the bath what are the other maintenance recommendations?
Once a month the water bath and the degassing lines should be rinsed with a 10% bleach solution. Fill the bath with the above solution, lower the transducer and run the degassing pump for a few minutes.
Raise the transducer and empty the tank. Lower the transducer and run the pump for 5-10 sec to empty the lines. Fill the water bath with fresh water and repeat the procedure.

12.
I have the Covaris Water Conditioning System. What kind of the maintenance should I perform?
The Water Conditioning System (WCS) automatically circulates water through a particulate filter and ultraviolent (UV) sterilizer to ensure that water remains clean and free of algae growth for up to one month. When the system runs continuously, it maintains the proper water quality required for optimum performance of the transducer. Without the WCS, proper water quality is maintained by daily changes of the water in the water bath. Once a month the water tank should be cleaned with a bleach solution as described above.
IMPORTANT: disconnect the hoses to and from the WCS when performing the bleaching procedure.
DO NOT circulate bleach solution through the WCS.
IMPORTANT: as the system is constantly circulating water in an open environment, evaporation is accelerated; this requires periodic replenishment of water. The frequency is dependent on the environment of the WCS.
13.
Why is it so important to degas the water in the tank?
Water in the tank (not in the chiller) serves as a coupler in the acoustic circuit. The need for clean and degassed water cannot be overstated. Poor water quality may dramatically impede the high frequency energy transfer. Insufficiently degassed water will readily scatter acoustic energy. The same can happen when water is contaminated with small particles. Keep in mind that at high frequency the acoustic energy may be scattered by dissolved gases and vapors present in water at standard atmospheric conditions as well as by particles suspended in water.
14.
Can I add algaecide to my water bath?
No. algaecide should not be added to the water in the tank to avoid formation of the aerosol containing algaecide during the AFA treatment. However, you can add algaecide to the water in the chiller since the water is circulating through a stainless steel loop immersed in the water bath.
15.
When analyzing DNA fragments on the BioAnalyzer I am noticing sometimes tailing or even a split peak. What is the probable cause?
Loading too much DNA on the chip can distort the peak causing split or tailing. The split peak or shoulder on the right hand side of the peak can also be caused by an occasional formation of an air gap in a microTUBE. This will result in partitioning of the liquid within the tube and thus the sample will not receive uniform acoustic treatment. As a result larger DNA fragments are observed in the upper part.
To avoid the air gap formation the microTUBE should be filed with 130µl of sample and care should be taken to avoid introduction of air during pipetting.
You can also use Covaris micro Tube Centrifuge Adaptors (P/N: 520059) to remove any air bubbles introduced during pipetting of the sample into the microTUBES. This reusable adapter fits most bench top microcentrifuges. The microTUBE should be centrifuged at around 1000 G for 20 sec.
16.
When analyzing DNA fragments on the BioAnalyzer I am noticing a small peak near the lower marker.
What is probable cause?
The extra peak can be caused by the presence of RNA in the DNA preparation. The RNA can be sheared similar to DNA into small fragments. However the single stranded RNA run faster than DNA fragment of the same length.

17.
Can I use the Covaris instrument to fragment RNA? If so, what conditions should I use?
Yes, it possible to fragment RNA using Covaris AFA technology. To fragment poly(A)mRNA into
200nt fragments we recommend the following conditions: 10% Duty Cycle, 5 Intensity, 200 cycles per burst and treatment time 60-90 sec. To fragment total RNA to 200 nt the same conditions can be used, however the treatment time is between 240 and 300 sec.
18.
We have 300bp DNA which we want to shear to 150-200bp. Can we use the recommended protocol?
In the case when the difference between the size of DNA starting material and desired fragment size is very small you need to increase the treatment time. We recommend testing the treatment time of 10 or even 14 min and checking the results on the BioAnalyzer.
19.
I am not getting expected DNA fragment sizes. How can I check whether the instrument is working properly?
To check whether the instrument is working properly we do recommend shearing a sample of genomic
DNA of known purity (for example, commercially available E. Coli or lambda DNA) using our recommended settings for the desired fragment size. If possible, run an aliquot on the BioAnalyzer DNA chip and compare the size distribution against our published protocols.
20.
I have an Illumina protocol for creating 400bp fragments that uses T6 round bottom glass tubes. I have only microTUBES. Can I use them instead and what conditions are recommended?
We do have a protocol for generating 400bp fragments size in micro TUBES published on the web site.
The conditions are as follows: 10% Duty Cycle, 4 Intensity, 200 cycles per burst, treatment time 55 sec.
21.
I am preparing shotgun library for the 454/Roche GS-FLX. I would like to use the Covaris instrument instead of a nebulizer. What conditions do you recommend?
Since the protocol calls for the DNA fragments in 500-800bp range you can use the conditions recommended for either 500 or 800bp fragments published here on the our web site.
22.
Do you provide DNA shearing service?
Yes, we do. For more information please contact CustomerService@covarisinc.com
or talk with your local sales representative.
23.
I notice a fiber inside the microTUBES. What is the purpose of this fiber, and should I be worried about contamination of my samples from this fiber during treatment?
The fiber inside the microTUBES serves a dual purpose. The first is providing nucleation sites for inertial cavitation. Cavitation is a process where a bubble in the liquid rapidly collapses creating a microjet that fragments the DNA. The second purpose is to allow for efficient mixing of the sample during processing. The acoustic fiber is thoroughly cleaned during the manufacturing process before being inserted inside the microTUBES and is free from organic contaminations.
24.
What is the best way to load and unload the microTUBES from the microTUBE holder for the S220?

Holding the micro Tube holder in one hand press the metal plunger up and insert the micro TUBE between the plastic fingers of the holder. Make sure that the micro TUBE is seated firmly and aligned in a vertical position between the fingers. Check the tube for bubbles inside. If the bubbles are present briefly centrifuge the micro Tube to remove them. After the treatment remove holder from the S220.
Holding the micro Tube holder in one hand press the plunger up and using the fingers of the other hand push the tube out.
25.
What do the settings of Duty Cycle, Intensity, and Cycles per Burst represent?
Duty Factor (Duty Cycle): DC=t
1
/ t
2
where t
1
is the transducer “on time” and t
2
is the burst period (sum of on time plus off time). In other words, duty cycle is the percentage of time that the transducer is actually transmitting acoustic energy.
Intensity: Intensity is the amplitude of the sine waves and it is proportional to acoustic power, (for example, Intensity 2 delivers twice the acoustic power of Intensity 1). Acoustic power is proportional to the square of acoustic pressure (A)
Cycles per burst (CPB): The number of sinusoidal pressure waves in a burst. Higher CPB is used to grow the cavitation bubbles.
26.
I do not see any settings for the fragment size I am interested in, but I see settings for size ranges a bit over and a bit below what I am interested in. How can I optimize settings to get the size range of interest?
You should look at the settings that bracket the DNA fragment size of interest. Take the treatment conditions for the larger fragment size and increase the treatment time until you will get the desired size.
Conversely, you can take treatment conditions for the smaller fragment and decrease the treatment time.
27.
Why does the size distribution of fragments seem to get wider with increasing size range?
The scale on an electropherogram generated by the Agilent BioAnalyzer DNA chip is logarithmic, hence the shape of the peak. A peak at 2000bp, with the same base width in seconds as a peak at 500 bp, will cover much wider bp range because of the logarithmic scale. Below is a picture of electropherogram of the 500bp DNA fragment and the same data shown using a linear scale.

28.
Do I need to do size selection after shearing using your technology?
Size selection is not typically carried out at the 100, 150, and 200 bp fragmentation for the library preparation for the SOLiD , Illumina, as well as the Agilent SureSelect protocol. If desired the size selection can be carry out using gels and Ampure beads after adapter ligation. Roche 454 protocol calls for a size selection as a part of their library preparation prior to adapter ligation.
29.
Aside from the phosphodiester bonds, are there any other bonds broken during processing of my samples with the Covaris AFA?
To date there has not been a study to show the affect of acoustic fragmentation on the bonds within the
DNA molecule. Indirect evidence from billions of bases sequences so far indicate that to a great extent only phosphodiester bonds are broken during AFA fragmentation of DNA.
30.
Can I optimize microTUBE settings for volumes less than 130µl?
It depends what volume you have in mind. Lowering the volume to 100µl would not require any changes, however, on occasion you might encounter an air gap problem described in the answer to the question #10. Some of our customers are successfully shearing 50µl DNA samples in the microTUBES.
In this case you need to cut the recommended Duty Cycle in half and adjust the method by slightly increasing the treatment time.
31.
How does sample concentration affect processing settings and time?
Large amounts of DNA (more than 30µg in 130µl) may require longer treatment times to ensure a homogeneous shearing result. Also due to increase in viscosity higher concentration of DNA may require an increase in temperature to 20 °C

32.
The chiller is for some reason taking over an hour to chill the water batch on my S220. What could be the cause?
There could be several reasons why the chiller doesn’t efficiently cool the water bath. First, you should check the fittings – are they firmly connected? Are the hoses free of kinks? Are the hoses being pinched? Have the length of the hoses been increased? Are any particulates present in the chiller water that can cause clogging? Has somebody changed the chiller set point? Is the water (or fluid) level in the chiller adequate; low level results in inefficient chilling.
33.
I have a VWR chiller that came with the Covaris instrument. I can’t change the temperature on the chiller control panel. What should I do?
The reason why the current settings cannot be changed is the fact that the local lockout feature on the controller has been activated. The screen is reading LLo. To unlock the controls, press and hold the
Select/Set Knob for 10 sec. The screen will read CAn and the setting can now be changed.
34.
I am using a liquid handler to remove my samples from the 96 microTUBE rack, but I cannot seem to get the final 20-30ul of sample out. Do you have any suggestions on the type of pipette tips I could use to get to the bottom of the tubes?
When using the robotic liquid handler we strongly recommend use of the 96 microTUBE plate instead of the 96 crimp-cap microTUBE rack. The Beckman P250 pipette tips work very well in aspirating the entire volume of sample from crimped capped microTubes.
35.
Can I store my samples at -20 and -80 in the microTUBES?
We don’t recommend storing samples in the microTUBES. After the treatment samples should be transferred to microcentrifuge tubes for storage.
36.
When I check my sheared samples on a gel, the smear profile looks much different that the size range you indicate on your protocol. Why is that?
The smear profile on a gel is depended on the concentration of the loaded DNA sample, as well as staining technique that is used. Overloading the gel will give an impression of a wider distribution. Also, staining the gel after the electrophoresis will avoid the gradient formation of Ethidium Bromide.
37.
In what type of buffers can I shear my DNA?
We recommend Tris-EDTA pH 8.0 buffer (TE) or 10mM Tris-HCl pH 8.0 to shear the DNA.
38.
Does the DNA source have an effect on the shearing conditions and results?
The Covaris AFA random fragmentation is a DNA source independent process. The protocols on
Covaris web site were developed using lambda and E. coli genomic DNA. Larger or shorter length of starting material may require longer dose to ensure the desired fragments sizes.
39.
When I am done using the Covaris system, how should I properly shut it down?
To shut down the instrument, turn off degassing and raise the acoustic assembly to lift the transducer out of the water, and empty the water bath. Place the tank back on the instrument, and turn on the degass pump for 5 seconds to empty the remaining liquid dry it with a lint-free cloth and place it back into

system. The long-term submersion of the transducer in the water bath when not in use may result in permanent damage to the system.
40.
What is AFA-grade water?
It is water meeting AFA-grade specifications (ASTM Type III or ISO Grade 3) such as Covaris
#520101. AFA-grade water is required for Covaris Focused-ultrasonicators to properly complete the acoustic circuit.
41.
How do you determine the target Base Pair (Peak)?
Looking at the electropherogram generated by the Agilent BioAnalyzer with the 12k chip we determine the maximum amplitude of the curve. This value determines the peak size.