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DIAGENODE
BIORUPTOR
®
PLUS
USER MANUAL
Europe Diagenode sa
/ CHU - Tour GIGA - B34 - 3e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info
@
diagenode.com
Important comments about DNA shearing
The Diagenode ACT (Adaptative Cavitation Transfer technology) process is highly reproducible, however attention must
be paid to the following treatment attributes to ensure best results:
•
Tubes:
At present, the recommended tube vessels are the Diagenode’s Bioruptor
®
0.5 ml Microtubes for DNA Shearing
(Cat No. WA-004-0500). Pay attention not to damage the cap when closing the tubes since this could alter sonication
results.
•
Sample volume:
The recommended volume of the Diagenode’s Bioruptor
®
0.5 ml Microtubes for DNA Shearing (Cat
No. WA-004-0500) is 100 μl. When using lower volumes (eg. 50 μl), less reproducible results may be observed due to
an alteration of the ultrasonic waves distribution in the sample fluid; thus, reducing the efficiency of sonication which
may result in broader size distribution or larger peaks.
•
Sample concentration:
Diagenode recommends using a DNA concentration ranging between 1 and 20 ng/μl
(10 ng/μl recommended). Using larger concentration (eg. 50-100 ng/μl) may result in broader peaks or variable peak
distribution.
•
Sample preparation:
Sample viscosity may have a major impact on sonication results. Careful resuspension of DNA
sample is strongly recommended before sonication processing. Multiple pipetting and gentle vortexing followed by a
short centrifugation to recover sample volume at the bottom of the tube is therefore strongly recommended. Storing
DNA samples on ice during 10-15 minutes before sonication has also been shown to improve reproducibility.
•
DNA Qualtity:
DNA quality and quantity must be considered carefully since bad quality and quantity DNA may have
several impacts on sonication and next-gen sequencing downstream applications. First, DNA contamination (eg. from
superfluous nucleic acids such as RNA, small nucleic acid fragments, excess proteins, or other contaminating materials)
may interfere with DNA measurement method leading to incorrect DNA quantitation thus. Also contaminating RNA in
genomic DNA preparation might generate a biased fragment distribution profile on microfluidics-based platform (eg.
Agilent Bioanalyzer) or alter sonication effiency.
Therefore it is highly recommended to use only high quality DNA when sonicating DNA for next-gen sequencing
library preparation. The DNA sample to be processed should be highly pure, having an OD260/280 ratio of between
1.8 and 2.0, and should be as intact as possible. DNA extracted using standard techniques (eg. Proteinase K digested,
double phenol/chloform extraction, ethanol precipitated, treatment with RNase-DNase free enzymatic digestion to
remove contaminant RNA) or commercial spin-column based kits are recommended.
•
Water temperature:
Propagation of ultrasound in a liquid unavoidably produces heat that can ultimately alter DNA
sample (eg. by thermal denaturation). To ensure the best preservation of the sample, it is recommended to start the
sonication process with cold water in the sonication bath. During sonication, especially when doing long sonication
runs, the temperature must also be controlled.
Note:
The permanent installation of the Bioruptor
®
in a cold room is possible, although not sufficient to avoid the
temperature increase due to sonication. This location would only replace the “pre-cooling” step described above.
•
Automatic temperature control:
A recirculating water cooler is used to guarantee the automatic temperature control
of the sonication bath during the whole sonication process. This water cooler (cat No. BioAcc-cool) produces a regular
water flow with a constant water level in the tank.
•
Sonication time:
Minor adjustments in cycle number may be made to optimize results for various sample types
and concentrations. The table above listing the cycle parameters and numbers is a recommended guideline. Actual
results may vary depending on the amount and type of starting material, concentration, viscosity and/or plastic tubes.
Diagenode recommends setting up a time dose response experiment for determining appropriate cycle number.
Larger length starting material (e.g. total genomic DNA) and higher concentration may require a longer dose to
ensure a homogeneous shearing result.
•
Sonication bath:
The sonication sonication bath is a critical component of the Bioruptor
®
sonication system.
1.
Water purity:
Contaminants such as algae and particules may alter the ultrasonic waves propagation,
