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TaqMAN PROBE qRT-PCR Master
.
Cat no.
W2701
W2701-8
Description
Recommended Protocol
Package
1 ml
8 X 1 ml
TaqMAN PROBE qRT­PCR Master mix is an optimised ready-to-use
solution for one-step quantitative RT-PCR assays. The master mix is
recommended for use with Labeled Fluorescent Probes, e.g. for
5’-Nuclease Assays or Hybridization probes. It comprises all the
components necessary to perform qRT-PCR: MMLV RTase (RNase H-),
antibody based hot-tart Taq DNA Polymerase, ultrapure dNTPs and
MgCl2. The user simply needs to add water, template and primers. An
enhanced buffer allows for RT reaction temperatures up to 50°C. This
can improve detection of more difficult targets as higher RT temperatures reduce nonspecific priming and facilitate melting of RNA secondary structures. The kit includes the components necessary for performing RT-PCR amplification, and have been successfully used to DNA
amplify and detect a variety of RNA targets. The kit provides a one-step,
simple, robust, inexpensive assay for detection and quantitative
analysis of gene expression directly from cells or RNA with intercalator
format.
Kit Contents
Contents
TaqMAN PROBE qRT-PCR Master
Cat.#
FS-RT-1072
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 TaqMAN PROBE qRT­PCR Master
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.
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:
Reaction Conditions
20 μl reaction
Component
10 μl
Final Conc.
1X
10μM Forward Primer
0.2~2.0 μl
0.1~1.0 μM
10μM Reverse Primer
0.2~2.0 μl
0.1~1.0 μM
PROBE qRT-PCR Master
Fluorescence Probe
Variable
0.1~1.0 μM
100 RX
Template DNA
Variable
≤ 500 ng / reaction
500 RX
Water, RNase-Free
up to 20 μl
Sizes
1 ml =(100 reactions)
NA
NOTE: In general, use greater than 0.5 μM primers for sensitivity and
less than 0.5 μM for specificity.
Applications
•
•
•
•
For in vitro research use only
Real-Time RT-PCR
Gene expression profiling
Gene knockdown verification
Array Validation
Quality Control
No endonuclease activity, nicking activity, exonuclease activity, or
priming activity has been detected.
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 at the reverse
transcription temperature for best results. The following table shows
recommended cycling conditions:
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.
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.
Note
Do not contaminate the Taq MAN PROBE qRT­PCR Master mix with
primers and template RNA used in individual reactions. Thaw and mix all
components thoroughly, spin down shortly and chill on ice.
One-step Real-time RT-PCR Conditions
Temp ( °C)
Time
Reverse Transcription 42 ~50
10 ~ 30 min.
Initial Denaturation
95
5 min.
Denature
10 ~ 30 sec.
95
Anneal (Detection) 55 ~68
10 ~ 60 sec.
Step
Cycle
1
1
20 ~ 40
NOTE: Cycling conditions may need to be optimized, depending on
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.
.
Quality Authorized by :
Jamie Ahn
1/3
For Research Use Only
FISHER MOLECULAR BIOLOGY
36 Terry Drive
.
Ver/
Trevose, PA 19048 - USA
PCR Instruments
Recommended ROX
concentration
Amount of ROX per 50 ul Reaction
BioRad: iCycler, MyiQ, MiQ 2, iQ 5
CFX-96, CFX-384, MJ Opticon , Opticon 2,
Chromo4, Mini Opticon
Qiagen: Rotor-Gene Q , Rotor Gene 3000 ,
Rotor Gene 6000
No Rox
Eppendorf: Mastercycler realplex
Illumina: Eco Real Time PCR System
Cepheid: Smart Cycler
Roche: LightCycler 480, LightCycler 2.0
ABI 7500, ABI 7500 Fast,
Stratagene MX4000P, MX3000P, MX3005P
ABI 7000, 7300, 7700, 7900HT and 7900HT
Fast
Low Rox * (0.1X)
Amount per 50 ul reaction:
0.1 ul (0.06-0.1ul)
Rox final conc.: 50 nM (30-50nM)
High Rox (1X)
Amount per 50 ul reaction:
1.0 ul (0.6-1.0ul)
Rox final Conc.:500nM (300-500 nM)
*Dilute 1X Rox 1:10 with ddH2O to obtain 0.1X Rox
2/3
FISHER MOLECULAR BIOLOGY
36 Terry Drive
Trevose, PA 19048 - USA
For Research Use Only
TaqMAN PROBE qRT­PCR Master
“REAL” One­step : cDNA synthesis and PCR performed simultaneously to provide fast and reliable results
Reay­to­use : No need premix preparation (mix enzyme with buffer) step, just add template RNA, primer and D.W
Easy and simple protocol to provide highly reproducibility and convenience
Performance : Thermostable RTase provide to cDNA synthesis over 50°C
Specificity : Minimize non­specific amplification and primer dimer by Reversible Hot­start (RHS™) Technology
Also can be obtained the best results by the Smart Buffer technology
Sensitivity : To maximize sensitivity capable of detecting low copy number target
Wide Application : Compatible with most Real­time PCR equipment
Higher Performance
Equal or superior results than major company's products
Fisher Molecular Biology
Bio-Rad
Excellent Reproducibility
Excellent dynamic range and reproducibility is
optimized for molecular diagnosis.
Fisher Molecular Biology
Roche
Excellent Stability
Performed RT-PCR reaction after 15 times freeze/thaw cycle,
no significant effect on performance was observed.
Stability of TaqMAN PROBE qRT­PCR Master through multiple freeze­thaw
events. Freeze­thaw events were performed by allowing qRT­PCR Master
to thaw from –20ºC to room temperature. Influenza A virus RNA was
amplified using the qRT­PCR Master after 15 times freeze­thaw cycles.
One­step Real­time RT­PCR reaction using the TaqMAN PROBE qRT­PCR
Master Kit. Human glyceraldehyde­3­phosphate dehydrogenase (GAPDH)
was detected in total RNA samples (n=2) ranging from 100 ng to 10 fg on the
Bio­Rad CFX96 System.
3/3
For Research Use Only
FISHER MOLECULAR BIOLOGY
36 Terry Drive
Trevose, PA 19048 - USA