Fragment FFPE DNA

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TEB (TE Buffer, user-supplied)

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Covaris 8 microTUBE strip with foil seals (if using LE220-plus Covaris instrument).

•

96-well PCR plate, semiskirted

•

Covaris 8 microTUBE strip with sample rack and waveguide (if using ME220 Covaris instrument).

•

96 microTUBE plate with SonoLab 10.0.1 (or higher), black (PN 520078 if using R230 Covaris instrument)

1.

Dilute each FFPE DNA sample with TEB as follows:

•

For 40 ng input, prepare 53 μl at 0.89 ng/µl. The assay requires a minimum extraction concentration of 3.93 ng/μl to allow for at least 41 µl TEB of the 53 µl volume.

2.

Pipette each diluted FFPE DNA sample 10 times to mix.

3.

Transfer 52 μl of each diluted FFPE DNA sample into a separate well of the ultrasonicator tube. Load the FFPE DNA into Covaris tube slowly to avoid creating bubbles.

4.

Fill any unused Covaris 8 microTUBE Strip wells with 52 μl TEB.

5.

Seal the microtube Strip with the foil seal.

6.

Centrifuge briefly.

7.

If using the Covaris LE220-plus, ME220 or R230 model, fragment the DNA using the following settings.

Setting	LE220-plus	ME220	R230
Peak incident power	450 watts	50 watts	450 watts
Duty Factor	30%	30%	25%
Cycles per burst	200	1000	600
Treatment time	250 seconds	10 seconds	—
Temperature	7°C	12°C	10°C
Pulse repeats	—	20	—
Average power	—	15 watts	—
Other	—	Wave guide	—
Iterations	—	—	20
Repeat Process Treatment Duration	—	—	10 seconds
Delay Duration	—	—	10 seconds
Dithering	—	—	1.5 mm @ 10 mm/s

8.

After sonication, centrifuge Covaris 8 microTUBE Strip briefly to collect droplets.

9.

Transfer 45 µl each sheared FFPE DNA sample to the corresponding well of the LP plate.