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Performing Digital PCR Hands-On

Each digital PCR platform has its own hands-on protocol. In this item, we have chosen to describe protocols used on the Naica™ System and on the QX200™ Droplet Digital™ PCR System.

For both systems, we will assume that you have already prepared your PCR mix and divided it into 8 tubes: one per sample. You have also loaded your 8 samples.

1. Naica™ System:

Since the Sapphire chips are already prefilled with oil, you have only one last step before launching the PCR reaction.

To do so, you will need :

  • 2 Sapphire Chips
  • tips and pipette adapted for 25 µL
  • 8 tall white caps (provided in the Sapphire Chips box)

Ready? Let’s go!

  1.  Unpack the Sapphire Chips and remove the white caps
  2. Gently pipette your 25 µl of PCR mix containing your samples into each inlet port
  3. Finally, seal each inlet port with the tall white caps

The hands-on part is done and you are now ready to place your Sapphire Chips into the Naica™ Geode where the partition generation and the thermal cycling will occur.

A short movie of the workflow is also available here!

2. QX200™ Droplet Digital™ PCR System:

Here, we describe the manual protocol. A faster way to proceed would be to use the QX200™ AutoDG™ Droplet Digital™ PCR System where the following steps are automated and integrated.

Two more steps before launching the PCR reaction :

A. Partitions generation

To do so, you will need :

  • 1 x DG8™ Cartridges for QX200™/ QX100™ Droplet Generator
  • 1x DG8™ Cartridges holder for QX200™/ QX100™ Droplet Generator
  • 1 x DG8™ Gasket for QX200™/ QX100™ Droplet Generator
  • 1 x QX200™ droplet generator
  • 1x Droplet Generation Oil for Probes
  • Rainin pipettes and tips adapted for 20µL and 70 µL

Ready? Let’s go!

  1.  Take the DG8™ Cartridges holder for QX200™/ QX100™ Droplet Generator. Open it by pressing the latches in the middle and sliding the two parts. Place one cartridge inside. Slide again in the original position to close
  2. Pipette your 20µL of PCR mix containing your sample in the middle wells of the cartridge. Then, pipette 70µL of droplet generation oil in the bottom wells
  3. Close the cartridge with one dedicated gasket
  4. Open the QX200™ Droplet generator by pressing the button on the top. Place the cartridge holder and close the droplet generator by pressing again the button

Partitions are now generated! The second and last hands-on part starts now :

B. Partitions transfer

To do so, you will need:

  • 1 x ddPCR 96-Well Plate
  • 1 x Pierceable foil plate seal
  • 1 x PCR plate sealer
  • Rainin pipettes and tips adapted for 40 µL

Let’s finish!

  1. Recover the DG8™ Cartridges holder from the Droplet Generator and remove the gasket. Do not open the cartridge holder.
  2. Gently pipette the 40µL of generated emulsion (top wells of the cartridge) and smoothly deliver it into a ddPCR 96-Well plate.
  3. Place the Pierceable foil on the ddPCR 96-well plate and proceed to the sealing part in the PCR plate sealer.
  4. Finally, place your ddPCR 96-well plate in the thermal cycler and launch your ddPCR program.