Peter MacCallum Cancer Centre Locations

305 Grattan Street Melbourne

Melbourne Victoria Bendigo
3000 Australia Box Hill
Moorabbin
Postal Address Sunshine
Locked Bag 1 ABeckett Street
Victoria 8006 Australia

Phone +61 3 8559 5000

Fax +61 3 03 8559 7379
ABN 42 100 504 883
petermac.org

Molecular Biomarkers & Translational Genomics Laboratory

Peter MacCallum Cancer Centre
Updated: August 2016

BioRad Digital Droplet PCR

Requirements:
- BioRad Master mix
o 2x QX200 ddPCR EvaGreen Supermix
OR
o 2x ddPCR Supermix for Probes
- RNase/DNase free water
- Assay primers
- Droplet cartridges
- Eppendorf Twin.tec 96 well plates
- Cooling block
- Validated pipette tips
- Droplet generator oil
o EvaGreen
OR
o Probe based
- Waste tip bucket
- Heat seal foil
- Droplet reader oil
- QuantaLife Software

NOTE: Samples run on the Droplet generator must be run in batches of 8. This is necessary for vacuum pressure.

A. Master Mix
1. Bring all reagents needed to room temperature.

2. Set up master mix reactions in Eppendorf twin.tec 96 well plate:

NOTE: Master mix reactions need to be at 25ul. Droplet generator will pipette out 20ul to mix with oil.

EvaGreen based reactions

Component Volume per Reaction (ul) Final Concentration
2x QX200 ddPCR EvaGreen Supermix 12.5 1x
Forward primer Variable 100-250nM
Reverse primer Variable 100-250nM
DNA template Variable 50fg to 100ng
RNase/DNase free water Variable
TOTAL 25ul

Patron: The Honourable Linda Dessau, AM Governor of Victoria

Probe based reactions
Component Volume per Reaction (ul) Final Concentration
2x ddPCR Supermix for Probes 12.5 1x
20x WT Primer/probe 1.25 900nM/250nM
20x Mut Primer/probe 1.25 900nM/250nM
Sample Variable 50fg to 100ng
RNase/DNase free water Variable
TOTAL 25ul

NOTE: Ensure there are no bubbles, as this will interfere with the generator.

B. Droplet generator
1. Turn on DG from powerpoint

2. DG interface will ask which Droplet Generator oil is being used.

Select appropriate Droplet Generator Oil.
Ensure correct oil is fitted.
Press OK.

3. On the DG interface, select Configure Plate

Highlight rows on 96 well plate containing sample.
Give experiment name.
The Droplet Generator will then instruct quantity of consumables needed.

4. Stock Droplet generator accordingly:

*Stock the machine from inside out to avoid contamination*

Stock in this order:

- Droplet Generator Oil
- Droplet cartridges
- Tips (without lid)
- Cooling block (purple when cold) (Should be kept at -20 between runs - upside down)
- An empty twin.tec plate for sample collection placed in the cooling block
- Sample plate (remove plate cover)

2
Peter MacCallum Cancer Centre Locations
305 Grattan Street Melbourne
Melbourne Victoria Bendigo
3000 Australia Box Hill
Moorabbin
Postal Address Sunshine
Locked Bag 1 ABeckett Street
Victoria 8006 Australia

Phone +61 3 8559 5000

Fax +61 3 03 8559 7379
ABN 42 100 504 883
petermac.org

NOTE: Generator cannot detect fresh plate in cold block. Ensure this is in place.

5. Press START Droplet Generation

Patron: The Honourable Linda Dessau, AM Governor of Victoria

Run: 45 mins for an entire 96 well plate.
Generator will collect 20ul of sample, combine with 70ul of oil and place into cartridge for partitioning.
Sample will then be deposited in new twin.tec plate sitting in cool block.

NOTE: Samples must be placed onto thermo cycler immediately after Droplet Generator. After thermo cycling,
samples can be left at 4 degrees (overnight if necessary) before reading.

C. Heat seal
1. Turn unit on
Machine will alert you to ensure block is outside the unit
2. Wait for unit to reach 180 degrees
3. Open machine door and place silver block inside
4. Place cold twin.tec plate in block
5. Place foil onto of plate, red line at the top
6. Close door
7. Press SEAL
Will heat plate to 180 degrees for 5 seconds
8. Move plate to thermo cycler
9. Remove block from unit and turn off machine.

D. Thermo cycler
Any thermo cycler can be used with necessary cycling conditions.

1. Place plate into thermo cycler and close the lid.

2. Select appropriate program:

EvaGreen based reactions

Cycling step Temperature Time Ramp Rate # Cycles
Enzyme activation 95 5 min 1
Denaturation 95 30 sec
Annealing/Extension 60 1 min 2 degrees/ sec 40
Signal 4 5 min 1
stabilization 90 5 min 1
Hold 4 Infinite 1

Probe based reactions

Cycling step Temperature Time Ramp Rate # Cycles
Enzyme activation 95 10 min 1
Denaturation 94 30 sec 40
Annealing/Extension 60 1 min 2 degrees/ sec 40
Enzyme deactivation 98 10 min 1
Hold 4 Infinite 1

Run: Approximately 2 hours.

4
Peter MacCallum Cancer Centre Locations
305 Grattan Street Melbourne
Melbourne Victoria Bendigo
3000 Australia Box Hill
Moorabbin
Postal Address Sunshine
Locked Bag 1 ABeckett Street
Victoria 8006 Australia

Phone +61 3 8559 5000

Fax +61 3 03 8559 7379
ABN 42 100 504 883
petermac.org

E. Droplet reader
1. Turn on Droplet Reader (before software).

2. Ensure there is enough Reader Oil. Empty waste container and place bleach inside.

3. Turn on laptop and open QuantaLife software.

Panel in the top right of the software should open.

4. Prime machine if has been off.

Flush machine between runs.

5. Open the machine

4. Remove the plate and adaptor
5. Place sample plate in with seal on
6. Place adaptor back on top
7. Close

8. Set up run in QuantaLife

- Select New (or Load and old template)
- Select analysis method (ie. Absolute, Rare Event detection or CNV)
- Select New
- Select Apply. Select OK.

- Enter plate name

- Highlight wells containing sample
- In the Experiment drop down menu, select analysis (ie. Absolute, Rare Event detection or CNV). Select
Apply
- Select Chemistry (EvaGreen/Probe based). Select Apply.
- In the Type drop down menu Select Ch1 Unknown. Select Apply.
- Select RUN in the left hand side panel
*Channel 1: The FAM channel. Channel 2: VIC or HEX*

- In the new window select Acquire data by Column

- Ensure correct chemistry is selected
- Select OK

Run: Approximately 1.5 mins per well.

Patron: The Honourable Linda Dessau, AM Governor of Victoria