In which order I should add the following component for PCR reaction using ddPCR?
Water
2x MasterMix
20x FAM
20x VIC
digested DNA
ddPCR reactions were assembled using standard protocols:
ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories)
was combined with 5–125 ng of sample, 1 µl of 20x assay
mix (1x primers at 900 nM each, 1x probes at 250 nM each),
4 units of restriction enzyme (HaeIII for RBM20 or MseI for
GRN), and water for a fnal reaction volume of 20 µl. Reactions
were converted into approximately 20,000 one-nanoliter
droplets using the QX200 Droplet Generator and transferred
to a 96-well plate for thermal cycling per manufacturer
recommendation for this supermix. For GRN assays, a discrete
2 minute extension step at 72°C was included, generating
a three-step thermal cycling protocol that accommodates a
longer amplicon (252 bp). After thermal cycling, droplets were
read on the QX200 Droplet Reader and assigned as positive
or negative based on fluorescence amplitude.
Hi Siti,
I use this order: 1. H2O, 2. Master mix, 3. primers - pipetting under the surface, 4. To close negative control, 5. DNA.
Regards,
Marcela
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