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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.
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.
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.
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
36)
37)
38)
39)
40) On the 'Dye Set' field Choose with probe reporters to use.
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.
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)
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