ddPCR sample prep and workflow

Question 1

What are some considerations for DNA pre-digestion?

Answer 1

No cut sites within the amplicon.
Wide range of enzymes with 4bp and 6bp recognitions sites work; methylation-insensitive enzymes are best.
RE buffers can cause problems, so digest in smallest volume possible then dilute and add to reaction mix.
Digestion particularly important above 75 ng per 25 uL reaction; not important for cDNA.

Question 2

How much DNA should be added to a ddPCR reaction?

Answer 2

Do not add more than 1 ug digested DNA. The highest confidence data is achieved at 1-2 CPD; approx 30,000 - 60,000 copies per 25 uL reaction. Assuming one copy per haploid genome, this is 100 ng - 200 ng human genomic DNA. for other species see application guide for link to database of genome sizes (DTU).

Question 3

How do you use the QX200 DG (manual DG) to generate droplets?

Answer 3

1. Ensure PCR reaction mix is very well mixed (recommend pipetting entire volume up and down >10 times or vortex–>spin being careful of aerosols).
2. Pre-load the DG8 cartridge into the cartridge holder
3. Transfer 20 uL PCR reaction mix to middle rows.
4. Transfer 70 uL droplet generation oil into bottom wells.
5. Attach gasket (ensuring it is held properly on each four corners).
6. Load into the DG and close the door.