Standard protocols for qPCR / qRT-PCR /RT-qPCR

You can use our qPCR Calculator tool to determine the required amount of reagents and dilutions for your assay.

Our qPCR Calculator – Calculate your assay reagents the easy way

qPCR Master Mix – 96-Well

Reagent 25 μl reaction volume final concentration
2x PCR Mastermix 12,5 µl 1 x
Forward primer (i.e. 5 pmol/µl) variable (i.e. 2 µl) 0.1 – 0.2 µM    
Reverse primer (i.e. 5 pmol/µl) variable (i.e. 2 µl) 0.1 – 0.2 µM    
Template DNA variable 0.1 – 10 ng/reaction
ddH2O fill up to 25 μl final volume per reaction

qPCR Master Mix – 384-Well

Reagent 10 μl reaction final concentration
2x PCR Mastermix 5 µl 1 x
Forward primer (i.e. 5 pmol/µl) variable (i.e. 1 µl) 0.1 – 0.2 µM    
Reverse primer (i.e. 5 pmol/µl) variable (i.e. 1 µl) 0.1 – 0.2 µM    
Template DNA variable 0.1 – 10 ng/reaction
ddH2O fill up to 10 μl final reaction volume

qPCR – 2 Step Cycler Settings

Step Time Temperature
Denaturation 3-5 minutes 95°C
35 – 40 cycles
Denaturation 10 seconds 95°C
Annealing + Extension Variable, depends on amplicon and mastermix, typically 45-60 seconds 60° – 72°C (depends on primer)

qPCR – 3 Step Cycler Settings

Step Time Temperature
Denaturation 3-5 minutes 95°C
35 – 40 cycles
Denaturation 10 seconds 95°C
Annealing 10 seconds 60° – 72°C depends on primer)
Extension Variable, depends on amplicon length, typically between 30 -45 seconds 72°C

Your Product Specialist

Jan Winter

Product Manager Genomics

PCR, qPCR, Magnetic Beads & CRISPR/Cas9

+49 (152) 3361 2537

jw@steinbrenner-laborsysteme.de