Applied Biosystems 7500 - Page 47

Chapter 2 Design the Standard Curve Experiment
Define the Methods and Materials
23
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
Notes
Design
Guidelines
When you design your own standard curve experiment:
• Select Standard Curve as the quantitation method. The standard curve method is
used to determine the absolute target quantity in samples. When setting up your
reaction plate, the standard curve method requires targets, standards, and samples.
• Select the reagents you want to use:
– Select TaqMan
®
Reagents if you want to use TaqMan reagents to detect
amplification and quantify the amount of target in the samples. TaqMan reagents
consist of two primers and a TaqMan
®
probe. The primers are designed to amplify
the target. The TaqMan probe is designed to hybridize to the target and generate
fluorescence when the target is amplified.
– Select
SYBR
®
Green Reagents if you want to use SYBR Green reagents to detect
amplification and quantify the amount of target in the samples. SYBR Green
reagents consist of two primers and SYBR
®
Green dye. The primers are designed to
amplify the target. The SYBR Green dye generates fluorescence when it binds to
double-stranded DNA. SYBR Green dye is often part of the SYBR Green master
mix added to the reaction. If you use SYBR Green dye, select the
Include Melt
Curve
check box to perform melt curve analysis of the amplified target.
Note: Although you can use other fluorescence-based reagents on the 7500/7500 Fast
system, you must design your experiment using Advanced Setup instead of the Design
Wizard.
• Select the appropriate ramp speed for the instrument run:
–Select Fast (~ 40 minutes to complete a run) if you are using Fast reagents for
the PCR reactions.
–Select Standard (~ 2 hours to complete a run) if you are using standard
reagents for the PCR reactions.
• Select the appropriate PCR template:
–Select cDNA (complementary DNA) if you are performing 2-step RT-PCR, and
you have already performed reverse transcription to convert the RNA to cDNA.
You are adding complementary DNA to the PCR reactions.
–Select RNA if you are performing 1-step RT-PCR. You are adding total RNA or
mRNA to the PCR reactions.
Note: To use the Fast ramp speed with RNA templates, you must design your
experiment using Advanced Setup instead of the Design Wizard.
–Select gDNA (genomic DNA) if you have already extracted the gDNA from
tissue or sample. You are adding purified genomic DNA to the PCR reactions.