FS-T-1171 Sybr Green qPCR Master Mix

SYBR Green qPCR Master Mix
Recommended Protocol
Sybr Green qPCR Master MIX is an optimized ready-to-use solution
for real-time quantitative PCR assays, incorporating SYBR Green I dye.
It comprises all the components necessary to perform qPCR:
Taq DNA Polymerase, ultrapure dNTPs, MgCl2 and SYBR Green I dye
The user simply needs to add water, template and primers.
Hot start DNA Polymerase is activated by a 5 minutes incubation step at
95°C. This prevents extension of nonspecifically annealed primers and
primer-dimers formed at low temperatures during qPCR setup.
The kit includes the components necessary for performing PCR amplification, and have been successfully used to amplify and detect a variety
of DNA targets such as genomic DNA, cDNA and plasmid DNA.
1. Thaw reagents at room temperature. Mix thoroughly and then
place on ice immediately after thawing.
2. Assemble reaction tubes on ice whenever possible to avoid
premature, nonspecific polymerase activity.
3.The following table shows recommended component volumes:
500 RXs
Reaction Conditions
Kit Contents
Sybr Green qPCR Master MIX
Prior to the experiment, it is prudent to carefully optimize experiment
conditions and to include controls at every stage. See pre-protocol
considerations for details.
This standard protocol applies to a single reaction where only template,
primers, and water need to be added to the qPCR Master (SYBR) mix.
For multiple reactions, scale-up volume of reaction components
proportionally. All reagents should be thawed on ice, gently mixed and
briefly centrifuged before use.
1000 RXs
20 μl reaction
1 ml (100 reactions)
Sybr Green qPCR Master
10.0 μl
Final Conc.
10μM Forward Primer
0.2~2.0 μl
0.1~1.0 μM
10μM Reverse Primer
Template DNA
0.2~2.0 μl
0.1~1.0 μM
≤ 500 ng/reaction
Water, RNase-Free
up to 20 μl
Real-time PCR
Detection and quantification of DNA and cDNA targets
Gene expression profiling
Gene knockdown verification
Microbial detection
Viral load determination
Array validation
SNP genotyping
Storage Conditions
Upon receipt, store all components at –20°C.
Store the Master mix at 4°C after thawing for up to 6 months, depending
on the expiration date, without showing any reduction in performance.
Do not contaminate the Sybr Green PCR Master Mix with
primers and template DNA used in individual reactions. Thaw and mix
all components thoroughly, spin down shortly and chill on ice.
Use of the ROX Reference Dye
ROX reference dye is not included in this kit and may be added to
compensate for non-PCR related variations in fluorescence. Addition of
the reference dye is optional. Optimizing the ROX dye concentration
within the qPCR reaction is an important aspect of setup. Too much
ROX in the qPCR reaction will reduce background but also makes a low
target signal difficult to distinguish from background.
PCR INSTRUMENTS using Sybr Green qPCR Master Mix (No Rox):
NOTE: In general, use greater than 0.5 μM primers for sensitivity and
less than 0.5 μM for specificity.
NOTE: Recommended amount of template per PCR reaction:
- < 50 ng plasmid or
- < 500~1000ng genomic DNA or
- 2μl of a 100μl single plaque eluate or
- one single bacterial colony
4. Ensure reactions are mixed thoroughly by pipetting or gentle
vortexing followed by a brief spin in a microcentrifuge.
5. Optional-Overlay reactions with one-half volume PCR-grade
mineral oil when not using heated lid on thermal cycler.
6.Transfer tubes on ice into a thermal cycler pre-warmed. The following
table shows recommended cycling conditions:
PCR Conditions
Initial Denaturation
Temp (°C)
5 min.
10 ~ 30 sec.
55 ~ 68
10 ~ 60 sec.
Melting curve analysis
65 ~ 95
2~5 sec./step
25 ~ 40
NOTE: Cycling conditions may need to be optimized, depending on
BioRad: iCycler, MyiQ, MiQ 2, iQ 5, CFX-96, CFX-384, MJ Opticon,
Option2, Chromo4, MiniOpticon
Qiagen: Roto-Gene Q, Roto-Gene3000, Roto-Gene 6000
Eppendorf: Mastercycler realplex Illumina: Eco RealTime PCR System
Cepheid: SmartCyler
Roche: LightCycler 480,LightCycler 2.0
PCR INSTRUMENTS using Sybr Green qPCR Master Mix (With Rox):
ABI: 5700, 7000, 7300, 7500 Fast, 7700, 7900, 7900HT, Step One,
Step One Plus Stratagene: MX4000P, MX3000P, MX3005P
different primer and template combinations. For example, raise the
annealing temperature to prevent non-specific primer binding,
increase extension time to generate longer PCR products.
NOTE: Shorter annealing step time (<10sec.) can be used for
amplicon <100bp.
Quality Control Analysis:
PCR sensitivity and reproducibility assay
Sensitivity and reproducibility in real-time PCR are tested in parallel
reactions containing 10-fold dilutions of nucleic acid template.
Unit Characterization Assay
Sybr Green is a Trade mark of Invitrogen Inc.
Specific activity was measured using a 2-fold serial dilution method.
Dilutions of enzyme were made in 1X reaction buffer. Reactions were
incubated 10 minutes at 75°C, plunged on ice, and analyzed using the
method of Sambrook and Russell.
For Research Use Only
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