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Primer Optimization Using Temperature Gradient 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 assay optimization is to determine the optimum annealing temperature (Ta) of the primers by testing identical reactions containing a fixed primer concentration, across a range of annealing temperatures. This can be achieved if a qPCR instrument with a temperature gradient block is available. In the format presented in this protocol, primers are included at a final concentration of 450 nM, and the gradient is orientated across the X axis of the block such that all columns are subjected to the same Ta (i.e., 12 different temperatures). In other instruments, the gradient is down the column such that all rows have the same Ta (i.e., 8 different temperatures). The following protocol can be applied to either, albeit after minor modifications.


  • Quantitative PCR instrument with integrated gradient block control function
  • Laminar flow hood for PCR set up (optional)


  • cDNA diluted 1:5-1:10, gDNA 10ng, synthetic oligo (10,000 copies) or other suitable template for optimization.
  • KiCqStart SYBR® Green ReadyMix™ (Sigma KCQS00/KCQS01/KCQS02/KCQS03; instrument specific, see Table P17-42).
  • 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 concentrated stocks (10 μM working stocks: GOI).
    •    Custom oligos can be ordered at

Table P17-42. 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 random priming or oligo-dT priming method and diluted 1:10 for use, but any suitable, alternative template may be used.
  • All reactions are run in duplicate as technical replicates.
  • If using a PCR plate, follow a plate schematic (e.g., shown in Figure P14-19) to ensure that the reaction mix, samples
    and controls are added to the correct wells.


1.    Prepare a master mix for 56 reactions according to Table P14-35. Mix well, avoiding bubbles.


Table P14-35. Reaction Master Mix for Ta Optimization.


Reagents Volume (μL) per
Single 20 μL Reaction
Volume (μL) for
56 Reactions
KiCqStart SYBR® Green qPCR
ReadyMix 2×
10 560
Forward primer (10 μM) 0.9 50.4
Reverse primer (10 μM) 0.9 50.4
PCR grade water 4.2 235.2


2.    Remove 448 μL of master mix from step 1 (i.e., half ) into a separate tube for setting up the No Template Control (NTC)

3.    Add 112 μL of template to the remaining master mix from step 2. Set Template master mix on ice.

4.    Add 112 μL of water to the other half of the master mix from step 2. Set NTC master mix on ice.

5.    Aliquot 20 μL Template master mix from step 3 into two rows of the PCR plate labeled GOI.

6.    Aliquot 20 μL NTC master mix from step 4 into two rows of the PCR plate labeled NTC.

7.    Cover plates and label. (Make sure the labeling does not obscure instrument excitation/detection light path.)

8.    Run samples according to the three-step protocol below
       (Note: These conditions are specific for FAST cycling protocols) ensuring that the annealing temperature has been
       defined on a gradient between the lowest and highest that would be appropriate for the primers (example shows
       54–70 °C). Steps 1–3 are repeated through 40 cycles. A standard dissociation curve is run after amplification.

Table P14-36. FAST qPCR Cycling Conditions for Ta Optimization.


FAST Cycling Conditions Temp (°C) Time (sec)
Initial denaturation/Hot Start 95 30
Steps 1–3 are repeated through 40 cycles
Step 1 95 5
Step 2 54–70 (gradient) 15
Step 3 72 10

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

Figure P14-19. Plate Layout for Ta Optimization With Identical Ta for Each Column.

Plate Layout for Ta Optimization With Identical Ta for Each Column.

Note: The distribution of the samples and controls across the temperature gradient. If the instrument has a temperature gradient that varies vertically down the plate column, the samples and controls will need to be re-arranged accordingly.