Primer dimer formation

 

When SYBR® Green dye is used in real-time PCR, any primer dimers will cause higher background, and may lead to a generation of CT<40 for NTC samples. The primer dimer is not usually detected unless a dissociation profile is generated. Because the background is higher, large amounts of primer dimers may alter the CT of your experimental samples and change the expression level interpretation.

 

 

A SYBR® Green amplification plot prior to the dissociation curve. In this view the presence of primer dimers cannot be determined.

 

 

A dissociation curve following a SYBR® Green real-time reaction. The presence of primer dimers can be easily identified in the dissociation curve by the additional peak(s). The primer dimer peaks are usually seen at low melting temperatures.

 

 

 

Solution

 

  1. Optimize primer concentration using the following combinations:

     

  2. Reverse Primer (nM)

    Forward Primer (nM)

    50

    300

    900

    50

    50/50

    300/50

    900/50

    300

    50/300

    300/300

    900/300

    900

    50/900

    300/900

    900/900

     

  3. Use an amount of primer that produces no primer dimer and gives optimal amplification efficiency.