Sfeir Lab, Aleks Penev, 9/17/2014

ddPCR

Prepare cDNA using the SSIII system according to their protocol using the
maximum amount of starting RNA (5ug) in the reaction. Use either random
hexamer or oligo-dT primed reactions and DO NOT DILUTE THE END
PRODUCT. You need as much signal as possible to probe for hTERT.
Prepare samples in groups of 8 (due to the vacuum mechanism of the droplet
generator). If you cannot make a full group of 8, use supermix and water
mixture to match the viscosity of your other samples.
Each reaction should have 3 dilutions (1x, 10x, & 50x works best) and a nontemplate control (NTC).
If working with new probe/primer mixes, each probe should initially be tested
individually before multiplex reactions are set up.

Loading volume for droplet generation is 20ul, so preparing 22ul supermix will
account for pippetting error using the multichannel (making 24ul is a good idea if
this is new to you).
- No more than 10% of your reaction mixture can be cDNA solution to prevent
salt interference during droplet PCR.
24ul Rxn Mix:
12ul 2x ddPCR Supermix (no UTP) (contains all enzymes+factors, keep cold, but
avoid freeze-thaw)
1.2ul 20x FAM primer+probe mix
(1.2ul)
(20x HEX primer+probe mix) -> only if multiplexing reactions
1.2-2.4ul
cDNA
7.2 - 9.6ul ddH2O

It works best to combine all reagents besides the cDNA to create a mastermix that can be used for all dilutions and NTC's.
Load the master-mix onto the mixing plate first and then add the cDNA,
avoiding bubbles.
At this point, take prepared samples to PCR core (MSB 307) and use
multichannel pippette and tips from the core to avoid plastic fragments
clogging the droplet generator.
Mix wells using the multichannel, mixing by pumping about 80% of the way
to avoid bubbles (mix is very viscous, so be careful).
Load 20ul into the bottom corner of the sample wells in the droplet generator
cassette.
Load 70ul of oil for probes into the oil wells from a new disposable trough.

Place rubber gasket over cassette in holder and place into droplet generator.

Once generator is done, use new tips to gather oil emulsion (~40-50ul) and
place in reader tray (blue eppendorf brand). Repeat for all samples (in groups
of 8).
Try to keep filled wells covered while generating other droplets to avoid
contamination.
Handle emulsion gently to avoid "rain" caused by reaction droplets shearing
and aggregating due to turbulent pippetting.

Once all loaded, place plate in pre-heated plate sealer. Setting is 180 C for 5
seconds.
Sealer takes <5 minutes to warm up.
Make sure sealing foil is oriented "sticky side" down, with the stripe facing up.
Once sealed, transfer to PCR cycler. DDPCR.60 is the saved program based on
the protocol from Biorad site but custom settings can also be used.
Make sure the ramping temperature is set to 2C/second because droplet
emulsions do not mix as fast as normal PCR reactions, so time has to be
allowed for even heating.
Reaction time is 2 hours.
Once complete, move PCR plate to plate reader and ensure all wells as set to
ABS reading and sensing both FAM and HEX fluorophores.
Labels can be assigned before starting the read (like while the PCR is
finishing) or afterwards.
Reading takes 15 minutes per column, 3 hours for an entire plate.
Software will automatically gate positive vs. negative hits but it's usually best
to go reaction-by-reaction (groups of 4 wells - the 3 dilutions and the NTC
together) to set the gating for the group.