V7

Digital PCR protocol

1) Determine the number of samples and wells.
2) For each well:
component
Droplet PCR Supermix*
F primer - Target (10M)
R primer - Target (10M)
Probe - Target (10M)
F primer - Reference
(10M)**
R primer - Reference
(10M)**
Probe - Reference
(10M)**
DNA (10-66ng) + H2O

one sample

All the
samples+1

10l
1l (500nM in well)
1l (500nM in well)
0.5l (250nM in
well)
1l (500nM in well)
1l (500nM in well)
0.5l (250nM in
well)
5l (7.5l if not using
duplex)

Total

20l

* Bio-rad #186-3023. **For a duplex test with two different reporters (one
with FAM and the other with VIC/HEX), If using one set of peimers+probe
only, add 2.5l H2O instead.

3) Mix stock solution for all the samples, plus one sample extra,
without adding DNA and H2O.
4) Distribute15l mix in to each PCR tube (13.5l if not using
duplex).
5) Add H2O to each tube.
6) Add DNA sample to each tube.
7) Vortex.
8) Spin down, until there are no bubbles.
9) Enter the DG8 droplet generator cartridge (Bio-Rad #186-3008)
into the holder.

10) Transfer 20l to the middle lane of the droplet generator

cartridge, place the tip in a small angle at the side wall of the well, a
little above the bottom of the well and not tie to the side wall.
10a) In unused wells insert a 10l 2x buffer control (Bio-Rad
#1863052)+10l H2O, or Supermix+H2O.

11) Add 70l of DG oil (Bio-Rad #186-3005) to the bottom lane. Also
to not used wells.
12) Place the disposable rubber (Bio-Rad #186-3009) on the
cartridge.

13) Enter the cartridge into the QX100 droplet generator.

14) Check that the two lights on the left are on.
15) Close.
16) When the right light and arrows light are on, take out the
cartridge.

(If the orange light is on, try to take out and to enter the
cartridge again)

17) Using Rainin RTL200F tips, load slowly 40l of liquid with
bubbles from the cassette in to a PCR plate (eppendorf
#951020362). place the tip in an angle at the side wall of the well, a
little above the bottom of the well and not tie to the side wall.
* briefly - just load and dispense the liquid slowly in a small angle
without touching the well.

18) Place the plate in the sealer plate support block of the PX1 PCR
plate sealer.

19) Place aluminum seal (Bio-Rad # 1814040) on the plate with the
red line up.

20) Program the sealer to 4 sec, 180oC

21a) Press 'Seal'.

21b) Turn the plate 180o.
21c) Press 'Seal'.
22) Run the plate in a PCR machine:
95 C 10 min 1 cycle
(94 C 30 sec, 60 C 1min) - 40 cycles
98 C 10 min 1 cycle
4C up to 24 hours
Total PCR time - 75 Minutes.
23) Place plate in the plate holder

24) Insert the plate into the QX100 droplet reader.

* Plate can be used again if there are empty wells and if the inner
transparent plastic didn't come apart.

25) Important Turn the Droplet reader on before connecting to the

computer and opening the software.

26) Open QuantaLife Program > load an already saved plate

scheme.
Saved templates are usually saved at C:\QuantaLife\Templates
, Or Double click on the first well in the experiment to setup a new
plate.
(Password to laptop QuantaLife)
27) To set up new plate > Experiment > add experiment.

28) Enter sample name.

29) In the 'Name' field give the experiment a name.
30) In the 'type' field pick CNV, ABS, RED.

31) If doing CNV in the 'Reference copies' field enter 2 (the number
of copies in the reference gene).

32)
33)
34)
35)

In
In
In
In

the supermix field choose the supermix type.

Assay1 field enter the FAM probe name
assay2 field enter the VIC/HEX probe name.
the type field choose the sample type.

36)
37)
38)
39)

When done, click on 'OK'

In each well enter the well name, experiment and assays data.
When done, Save the plate scheme.
click on 'Run' to start the droplet reading from the plate.

40) On the 'Dye Set' field Choose with probe reporters to use.

41) Click 'OK' to start the reading.

42) When done, the results are saved at C:\QuantaLife\Data
43) Click on 'Analyze', or open a saved qlp results file.
44) If you didn't entered the experiment type (ABS/RED/CNV) or the
wells data correctly before the run, you can setup a new experiment
in the plate with the correct data, and hit 'analyze' again.
45) In the 1D icon you can see the droplets reading data.

46) The upper colorful circles represent the positive droplets, and
the lower black circles represent the negative droplets.
You can mark a single, or multiple wells.

45) You can setup manually for each well the threshold with above it
will be the positive droplets.

46) In 2D analyze you will see both channels data.

47) If the software could not determine the results automatically,

you will see a red 'CHECK' mark in the 'Status' field in the wells
description area in the upper left side of the screen. You can see the
reason for that if you stand with the mouse above it.

48) You can set the results for that well manually by setting the
threshold manually.
49) On the 'Copy number' field you will see the CNV results.
50) The order of the results in the graph is set by the wells picking
order.
Instructions videos for ddPCR technology:
http://www.youtube.com/watch?v=Qwma-1Ek-Y4
http://www.youtube.com/watch?
v=GB4wcQsCawU&list=PLA8F806265E6F59ED
Each nucleic molecule is separated inside a single oil bubble (Droplet) and
goes a PCR amplification process with a set of primers and fluorescent
.probes
Than each droplet measured to bubbles that are positive, Or negative to
that primers and probe set. Providing a positive\negative ratio, thus
providing absolute quantification

DNA Digestion:
DNA fragmentation by restriction enzymes may be use in order to
separates tandem gene copies, to reduce sample viscosity and to ensure
proper droplet formation and homogeneity.
Digest DNA with enzyme that does not cut inside the amplicon. Already
broken DNA samples like DNA from paraffin origin, might not need DNA
digestion.
Choose what enzyme to use with online tools that can predict restriction
areas in a sequence. like Nebcutter.
http://tools.neb.com/NEBcutter2/
Optimal annealing temperature for primers and probes:
Perform a preliminary temperature gradient test using a PCR machine.
In a PCR plate test different annealing temperatures in different wells, than
read plate in a Droplet reader and choose a temperature that has good
separation between positive and negative droplets and high positive
droplets formation.
In the sample below an annealing temperature of 60.5 oC gives the best
results.

Equipment
Filter tips: 10l short and long, 20l, 200l, 200l (Rainin).
Pipettors: <1l, 0.5-10l, 2-20l, 20-200l.
Microtubes (transparent): 200l, 700l.
Ultra pure water.
PCR strip caps.
DG oil (Bio-rad #186-3005)
Aluminum plate seal (Bio-rad # 1814040
Stored in -20oC:
Droplet supermix X2 (Bio-rad #186-3023)

Primers and probes.

dd-pcr Short Version protocol:
digital PCR
16ng of Genomic DNA samples were added to 2xddPCR supermix (Bio-rad)
together with a final concentration of 500nM of each primer and 250nM
probe in duplex of the tested gene and a control gene. RnaseP served as a
control reference gene. probes for the tested genes contained a FAM
reporter and RnaseP probes contained HEX. Samples were converted into
droplets using QX100 droplet generator (Bio-rad) and undergo thermal
cycles under the following conditions: 95 oC for 10min followed by 40
cycles of 94oC for 30 sec, 60oC for 60sec and one cycle of 98oC for 10 min
and 4oC for up to 24 hours. after PCR the plates were loaded into QX100
droplet reader (Bio-rad) and the value and the poisson distribution were
calculated using Quantasoft software (Bio-rad).

References
1) Chrissy h Roberts, Wei Jiang, Jyothi Jayaraman, John Trowsdale, Martin J
Holland and James A Traherne (2014). Killer-cell Immunoglobulin-like
Receptor gene linkage and copy number variation analysis by droplet
digital PCR. Genome Medicine, 6:20 doi:10.1186/gm537
2) Huggett, J. F., Foy, C. A., Benes, V., Emslie, K., Garson, J. A., Haynes, R.,
Bustin, S. A. (2013). The digital MIQE guidelines: Minimum Information
for Publication of Quantitative Digital PCR Experiments. Clinical chemistry,
59(6), 892902. doi:10.1373/clinchem.2013.206375

written by: Itai