Agilent Brilliant III Ultra-Fast QRT-PCR Master Mix User Guide

Brilliant III Ultra-Fast QRT-PCR Master Mix

Quick Reference Guide for the ABI StepOnePlus

Real-Time PCR System

This quick reference guide provides an optimized protocol for using the Stratagene Brilliant III Ultra- Fast QRT- PCR Master Mix with the StepOnePlus Real- Time PCR System from Applied Biosystems. For detailed instructions, refer to the full product manual.

Prepare the

Reactions

1 Dilute the reference dye 1:50 using nuclease- free PCR- grade water.

2 Prepare the experimental reactions by combining the components of the

reagent mixture in the order listed in the table below. Prepare a single reagent mixture for replicate reactions (plus at least one reaction volume excess) using multiples of each component. Keep reagent mixture on ice.

Reagent Mixture

Nuclease-free PCR-grade water to bring final volume to 20 μl (including RNA)

10 μl of 2× QRT-PCR Master Mix

x μl of experimental probe at optimized concentration (100–600 nM)

x μl of upstream primer at optimized concentration (200–600 nM)

x μl of downstream primer at optimized concentration (200–600 nM)

0.3 μl of diluted reference dye

0.2 μl of 100 mM DTT

1 μl of RT/RNase Block

3 Gently mix the reagent mixture without creating bubbles, then distribute

the mixture to the experimental reaction tubes. Keep the reactions on ice.

4 Add x μl of experimental RNA to each reaction to bring the final

reaction volume to 20 μl

for different RNA templates.

. The table below lists a suggested quantity range

RNA Quantity per reaction

Total RNA 0.1 pg – 100 ng

mRNA 0.1 pg – 1 ng

5 Mix the reactions without creating bubbles, then centrifuge briefly.

Stratagene Products 1

Agilent Technologies

Set Up the

QPCR Plate and

Thermal Profile

1 From the Home screen of the StepOnePlus software, click Advanced

Setup.

2 Complete the Setup screens for a new experiment as needed.

On the Experiment Properties screen, select TaqMan Reagents and the Fast ramp speed.

Run the PCR

Program

On the Run Method screen, set the reaction volume to 20

l and adjust the thermal profile according to the image below. Note that a new step needs to be added to the beginning of the profile for the 50°C incubation.

1 Place the reactions in the StepOnePlus instrument.

2 On the Run screen, click START RUN.

Analyze Data 1 Analyze the results of the run as needed for your experiment.

Notice to Purchaser

Practice of the patented 5´ Nuclease Process requires a license from Applied Biosystems. The purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research when used with the separate purchase of Licensed Probe. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Product Information

Catalog #600884, 400 reactions

Catalog #600885, 4000 reactions

*For other countries, please contact your local sales representative at www.agilent.com/chem/contactus

2 Brilliant III Ultra-Fast QRT-PCR Master Mix

Ordering Information

By phone (US only*): 800-424-5444, x3

On the web: www.stratagene.com

Technical Services

By phone (US only*): 800-894-1304, x2

By email: techservices@agilent.com