Reaction Conditions

Q: What is the reaction volume? 
A: qPCR is done in a 25 µl rxn made up of 12.5 µL of 2X Universal Master Mix II (Applied Biosystems), 7.5 µL of primers and probe, and 5 µL of template.  ddPCR is done in a 20 µL reaction consisting of 2X ddPCR PCR Master Mix (Bio-Rad), 1 µL 20X primer/probe mix, and 9 µL template.

Q: What is included in the qPCR mastermix? 
A: Platinum® Taq DNA Polymerase, ROX internal control, UDG (to prevent carryover contamination), dNTPs and buffers.

Q: What is the concentration of the samples used in the reaction ?
A: Most reactions are done at 10 ng/µL (based on the starting total RNA amount in the cDNA synthesis). However, this can vary depending on level of target expression and amount of sample provided. The concentration will always be included on the provided setup sheet.

Q: What is the concentration of the primers and probes? 
A: This will vary depending on what detection system you use. The classic TaqMan primers are usually at 900 nM and the probe at 200 nM. However, this can be re-optimized if needed. The concentrations will always be included on the provided setup sheet.  For ddPCR, the TaqMan primers are at 900 nM and probe at 250 nM.

Q: What are the thermal cycler conditions used? 
A: For qPCR, we perform a standard two-step PCR reaction.

• 50ºC for 2 min to get optimal UDG enzyme activity.
• 95ºC for 10 min to activate the DNA Polymerase.
• 45 cycles of 95ºC for 15 sec to denature the sample and 60ºC for 1 min for the annealing and extension.

For ddPCR, thermal cycling is done using a standard PCR block:

• 95ºC for 10 min
• 40 cycles of 94ºC 30 seconds, 60ºC 60 sec
• 98ºC 10 min
• hold 4ºC indefinitely

The cycles will be extended to 50 cycles for low expressing targets.