Primer Concentration Optimization Protocol

Optimization of qPCR Conditions

Optimization of qPCR conditions is important for the development of a robust assay. Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insensitive assays. The two main approaches are optimization of primer concentration and/or annealing temperatures.

One approach to optimizing primer concentrations is to create a matrix of reactions. This is used to test a range of concentrations for each primer against different concentrations of the partner primer. In the example provided in this protocol, a 6×6 matrix testing six concentrations (e.g., 50 nM to 800 nM) is demonstrated. The quantities stated in this protocol will allow each condition to be run in duplicate.


  • Quantitative PCR instrument
  • Laminar flow hood for PCR set up (optional)


  • Suitable assay template, e.g., cDNA diluted 1:10, gDNA, or synthetic oligo template.
  • KiCqStart SYBR® Green ReadyMix™ (Sigma KCQS00/KCQS01/KCQS02/KCQS03—depending on instrument, see Table P4-6 for instrument compatibility).
  • PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
  • Forward and reverse primers concentration stocks (10 μM working stocks).
    •    Custom oligos can be ordered at

Table P13-31. SYBR Green PCR Mix Selection Guide.

Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2)
KiCqStart® SYBR® Green qPCR ReadyMix™,
Cat. No. KCQS00
KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox ,
Cat. No. KCQS01
KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX,
Cat. No. KCQS02
KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ,
Cat. No. KCQS03
Compatible Instruments: Compatible Instruments: Compatible Instruments: Compatible Instruments:
Bio-Rad CFX384™ Applied Biosystems 7500 Applied Biosystems 5700 Bio-Rad iCycler iQ™
Bio-Rad CFX96™ Applied Biosystems 7500 Applied Biosystems 7000 Bio-Rad iQ™5
Bio-Rad MiniOpticon™ Fast Applied Biosystems ViiA 7 Applied Biosystems 7300 Bio-Rad MyiQ™
Bio-Rad MyiQ™ Stratagene Mx3000P® Applied Biosystems 7700  
Bio-Rad/MJ Chromo4™ Stratagene Mx3005P™ Applied Biosystems 7900  
Bio-Rad/MJ Opticon 2 Stratagene Mx4000™ Applied Biosystems 7900 HT Fast  
Bio-Rad/MJ Opticon®   Applied Biosystems 7900HT  
Cepheid SmartCycler®   Applied Biosystems StepOnePlus™  
Eppendorf Mastercycler® ep realplex   Applied Biosystems StepOne™  
Eppendorf Mastercycler® ep realplex2 s      
Illumina Eco qPCR      
Qiagen/Corbett Rotor-Gene® 3000      
Qiagen/Corbett Rotor-Gene® 6000      
Qiagen/Corbett Rotor-Gene® Q      
Roche LightCycler® 480      



Notes for this Protocol

  • cDNA is generated using a random primer or oligo-dT priming method and diluted 1:10 for use, but any suitable, alternative template may be used.
  • All samples are run in duplicate according to the plate layout (Figure P13-18).


Figure P13-18. Schematic Representation of the Primer Optimization Plate Layout.

Schematic Representation of the Primer Optimization Plate Layout.


Note: 2.0 μL of each primer will be added to the reaction of 20 μL total volume. For this reason, primer stocks are 10 times the required concentration to achieve the desired final concentration.

1.    Using the 10 μM primer stock, make a dilution of both primer stocks to 0.5, 1, 2, 4, 6 and 8 μM as shown in
       Table P13-32.

Table P13-32. Primer Dilution Scheme for Primer Concentration Optimization.


Concentration (μM)
Volume (μL)
Volume (μL)
10 μM Stock
Volume (μL)
0.5 47.5 2.5 50
1 45 5 50
2 40 10 50
4 30 20 50
6 20 30 50
8 20 80 100


2.    Prepare a qPCR master mix according to Table P13-33   
       (Note: Template and cDNA are added separately in step 5). Mix well.

Table P13-33. Reaction Master Mix for Primer Concentration Optimization.


Reagents Volume (μL) per
Single 20 μL Reaction
Volume (μL) for
Whole Plate
(84+10% pipetting error)    
2× KiCqStart SYBR® Green qPCR
10 924
PCR grade water 3 277.2
Reference dye (Optional) 1 92.4
Template, e.g., cDNA
(diluted 1:5 to 1:10 of stock)
2 *
Forward primer 2 *
Reverse primer 2 *

*Note: Do not add cDNA and primers until step 5.


3.    Remove 184.8 μL (for 12× NTC) of master mix from step 2 into a separate tube to use for setting up the
       No Template Control (NTC).

4.    Add 26.4 μL of dH2O to the NTC mix in step 3 (to replace template).
       Note: Set NTC mix on ice for later use.

5.    Add 158.4 μL of cDNA template to the remaining master mix from step 2. Set master mix on ice.

6.    Add 2.0 μL of appropriate reverse primer dilutions into the PCR plate according to Figure P13-18; also adding
       800 nM concentration to the NTC row.

7.    Add 2.0 μL of appropriate forward primer dilutions into the PCR plate according to Figure P13-18.

8.    Aliquot 16 μL master mix from step 5 into the PCR plate in the wells corresponding to test positions.

9.    Aliquot 16 μL master mix from step 4 into the PCR plate for NTC reactions.

10.  Seal plates and label. (Make sure labeling does not obscure instrument excitation/detection light path).

11.  Run samples according to the two-step protocol in Table P13-34. Steps 1 and 2 are repeated through 40 cycles
       and followed by a dissociation curve analysis.

Table P13-34. PCR Cycling Conditions for Primer Concentration Optimization.


Cycling Conditions Temp (°C) Time (sec)
Initial denaturation/Hot Start 95 30
Steps 1–2 are repeated through 40 Cycles
Step 1 95 5
Step 2 60 30

Note: Use standard dissociation curve protocol (data collection).