Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 Experimental Design for SYBR Green: based on relative standard curve method

Standard curve samples are typically serial diluted 5 fold at a time or 10 fold at a time. At minimum, five concentrations should be used for the standard curve and no fewer than three replicates for any concentration should be used.

Table1: A typical 96-well plate format for a SYBR Green assay utilizing relative standard curve method.

A typical experiment is designed with either the ABI 2x SYBR Green PCR Master Mix (ABI # 4309155) or the ABI SYBR Green Core Reagents Kit. For the SYBR Green PCR Master Mix, the following master mix is made and aliquated as such into a 96well plate.

Table 2: Typical Master Mix Set up for SYBR Green Master Mix.

The advantage to setting up a real time experiment with the SYBR Green Core Reagents Kit is you have the flexibility of controlling the concentration of certain components (such as magnesium concentration) within the master mix. Table 3 shows a typical set up for the core reagents kit.

Table 3: Typical Master Mix Set up for SYBR Green Core Reagents Kit.

 

Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 
 RRC Core Genomics Facility
University of Illinois at Chicago
2003