Protocol

DNA FRAGMENTATION

This section describes three methods for fragmentation of genomic DNA prior to ULS labeling.
Either of them can be used. Since the genomic DNA needs to be clean from salt and bivalent
cations like Mg2+ for ULS labeling, we recommend fragmentation protocols that do not need any
addition of reagents and that can be standardized easily.
Restriction enzyme digestion is not recommended for DNA fragmentation, as this is more time
consuming, and needs additional clean-up since enzyme buffers would slow down or inhibit
subsequent ULS labeling.

DNA quality: important notes

OD260/OD280 > 1.8 and OD260/OD230 > 1.8: to determine possible protein and salt contamination
If either of these values is <1.8, use a Silica clean-up column like the DNeasy blood and tissue kit
(Qiagen, cat# 69504), QIAamp DNA Micro Kit (#56304 Qiagen), DNA Clean & Concentrator™-5
(Zymo Research #D4003) or the Wizard SV gel and PCR cleanup system (Promega #A9282).
Important: In each of these methods (1) make sure to ADD an extra final washing step using
80% ethanol before the elution, and (2) elute in nuclease-free water (NOT in elution buffer,
because the EDTA present in most elution buffers could inhibit the ULS labeling reaction).

DNA integrity:
It is important that fragment sizes of test and referent samples are of similar size range.

For hybridization, the optimal DNA fragment length is 100-600 bp

MATERIALS

- For sonication: Bioruptor (Diagenode, Liege, Belgium) or small tip sonicator, e.g. Soniprep 150
(Sanyo MSE, London, UK) or Vibra-Cell (Sonics, Newtown CT, US).
- For heat fragmentation: PCR machine.
- 1% agarose gel (or Agilent 2100 Bioanalyzer)

PROTOCOLS

a. Sonication (Small tip protocol)

- Adjust sample volume with nuclease-free water to minimally 100 µl (minimal DNA
concentration: 20 ng/µl).
- Sonicate 5 x 1 min at 5 µm amplitude, while keeping the sample on ice. In between
sonication steps, leave the DNA on ice for 1 min.
- Check the fragment sizes of the genomic DNA on a 1% agarose gel. Fragment sizes
should be 100 – 600 bp. If not, sonicate again for 2 x 1 min.
b. Sonication: Bioruptor
- Pre-cool the water tank with ice cold water for 30 mins.
- Adjust sample volume with nuclease-free water to 100 µl in a 0.5 ml or 1.5 ml Eppendorf
tube. (minimal DNA concentration: 20 ng/µl)
- Fill the remaining positions with dummy Eppendorf tubes containing 100 µl of H2O.
- Sonicate the genomic DNA for 30 mins. (2 x 15 mins.) on “high”, 1mins. OFF, 1 mins.
ON.
- Check the fragment sizes of the genomic DNA on a 1% agarose gel. Fragment sizes
should be 100 – 600 bp. If not, sonicate the genomic DNA for an additional 30 mins. (2 x
15 mins.) on “high”, 1 mins. OFF, 1 mins. ON.

c. Heat fragmentation
- Use a sample volume of 20-100 μl
- Heat the sample 30mins. at 95°C in a PCR machine.
- Check the fragment sizes of the genomic DNA on a 1% agarose gel. Fragment sizes
should be 100 – 600 bp. If not, heat the sample again for 30 mins. at 95°C in a PCR
machine.

Heat fragmentation of genomic DNA. Intact DNA (>15kb, lane 2) was subjected to a time series of 95˚C
heat incubation and run on 1% agarose gel. Optimal fragment length for BAC microarray hybridization is
100-600bp. Heat incubation time to obtain such fragments might be dependent on quality of the sample:
increasing salt concentration lowers fragmentation speed.