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.

qPCR Master Mix – 96-Well

Reagent25 μl reaction volumefinal concentration
2x PCR Mastermix12,5 µl1 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 DNAvariable0.1 – 10 ng/reaction
ddH2Ofill up to 25 μl final volume per reaction

qPCR Master Mix – 384-Well

Reagent10 μl reactionfinal concentration
2x PCR Mastermix5 µl1 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 DNAvariable0.1 – 10 ng/reaction
ddH2Ofill up to 10 μl final reaction volume

qPCR – 2 Step Cycler Settings

StepTimeTemperature
Denaturation3-5 minutes95°C
35 – 40 cycles
Denaturation10 seconds95°C
Annealing + ExtensionVariable, depends on amplicon and mastermix, typically 45-60 seconds60° – 72°C (depends on primer)

qPCR – 3 Step Cycler Settings

StepTimeTemperature
Denaturation3-5 minutes95°C
35 – 40 cycles
Denaturation10 seconds95°C
Annealing10 seconds60° – 72°C depends on primer)
ExtensionVariable, depends on amplicon length, typically between 30 -45 seconds72°C

Your Product Specialist

Jan Winter

Product Manager Genomics

PCR, qPCR, Magnetic Beads & CRISPR/Cas9

+49 (152) 3361 2537

jw@steinbrenner-laborsysteme.de