How to optimize primer concentrations for SYBR qPCR

SYBR green assays suffer from an important artifact. Fluorescent detection cannot distinguish between the formation of PCR product and unspecific DNA synthesis. The most commonly observed unspecific PCR artifact is ‘primer dimer’, which results from self annealing and priming of primers during the annealing step of PCR. The following figure illustrates and example of primer dimer often observed in PCR reaction.

This is an example of primer dimer formed in a typical PCR reaction. (B) An example of a dilution series of a forward primer in four different PCR reactions (keeping the same concentration of reverse primer). Primer concentrations were (1) 500 nM, (2) 250 nM, (3) 125 nM, and (4) 67.5 nM. The reaction with 125nM forward primer has significantly less primer dimer, but maintains good amplification, while the reaction with 67.5nM forward primer displays loss of product formation.

To minimize the formation of primer dimer, a matrix of different primer concentrations is typically run for each new primer pair. The following matrix is commonly run in our lab:

• Make a 1 ng μl-1 positive control genomic DNA stock using a strain that contains your target gene.
• Dilute your primers to a concentration of 100 μM in DNA-free water.

For each of your primers :

• Add 2μl of primer to 200μl Invitrogen PCR Supermix to make a 1μM primer dilution in Supermix.
• Transfer 100μl of the 1μM primer dilution to a fresh tube containing 100μl Invitrogen PCR Supermix to make a 500nM primer dilution in mastermix.
• Transfer 100μl of the 500nM primer dilution to a fresh tube containing 100μl Invitrogen PCR Supermix to make a 250nM primer dilution in mastermix.
• Transfer 100μl of the 250nM primer dilution to a fresh tube containing 100 μl Invitrogen PCR Supermix to make a 125nM primer dilution in mastermix.

• Set up 16 PCR tubes and add the following to generate the concentration matrix shown above: (P1- Forward Primer; P2 – Reverse Primer)

• 1 – 20μl of 1μM P1 dilution; 20μl of 1μM P2 dilution
• 2 – 20μl of 1μM P1 dilution; 20μl of 500nM P2 dilution
• 3 – 20μl of 1μM P1 dilution; 20μl of 250nM P2 dilution
• 4 – 20μl of 1μM P1 dilution; 20μl of 125nM P2 dilution
• 5 – 20μl of 500nM P1 dilution; 20μl of 1μM P2 dilution
• 6 – 20μl of 500nM P1 dilution; 20μl of 500nM P2 dilution
• 7 – 20μl of 500nM P1 dilution; 20μl of 250nM P2 dilution
• 8 – 20μl of 500nM P1 dilution; 20μl of 125nM P2 dilution
• 9 – 20μl of 250nM P1 dilution; 20μl of 1μM P2 dilution
• 10 – 20μl of 250nM P1 dilution; 20μl of 500nM P2 dilution
• 11 – 20μl of 250nM P1 dilution; 20μl of 250nM P2 dilution
• 12 – 20μl of 250nM P1 dilution; 20μl of 125nM P2 dilution
• 13 – 20μl of 125nM P1 dilution; 20μl of 1μM P2 dilution
• 14 – 20μl of 125nM P1 dilution; 20μl of 500nM P2 dilution
• 15 – 20μl of 125nM P1 dilution; 20μl of 250nM P2 dilution
• 16 – 20μl of 125nM P1 dilution; 20μl of 125nM P2 dilution

• Add 2 μl of PCR positive control DNA to each tube
• Run PCR at desired conditions
• Run agarose gel using 5μl from each tube and find the combination that produces the least amount of primer dimer, while not showing a significant decrease of PCR product formation.