Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 

For SYBR Green based amplicon detection, it is important to run a dissociation curve following the real time PCR. This is due to the fact that SYBR Green will detect any double stranded DNA including primer dimers, contaminating DNA, and PCR product from misannealed primer. By viewing a dissociation curve, you ensure that the desired amplicon was detected. A typical plot of the dissociation curve is shown in Figure 3.

 

Figure 3: Melting Curve for Standard Curve Samples in Real Time for GAPDH

 

The Tm of the amplicon starts at the point of inflection of the melting curve profile. Fron Figure 3, we can see that the inflection point occurs at 82.5 degC for GAPDH.

It may be more helpful to look at the derivative plot of the melting curve depicted on Figure 4.

 

Figure 4: Derivative Melting Curve for Standard Curve Samples in Real Time, GAPDH Endogeneous Control

 

It is apparent that the point of infection (melting temperature of the amplicon) occurs at 82.5 degC. Also we can see that no contaminating products are present in this reaction. Contaminating DNA or primer dimers would show up as an additional peak separate from the desired amplicon peak.

     

Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

RRC Core Genomics Facility
University of Illinois at Chicago
2003