• Optimized for Real Time PCR with SYBR Green I
• Easy Preparation
• Increased Specificity

Description

SYBR^®Premix Ex Taq™ (Perfect Real Time) is a 2× concentration of premix reagent including TaKaRa Ex Taq™ HS and SYBR^® Green I^*1, specially designed for real-time PCR with intercalator method. This product combines the high performance of TaKaRa Ex Taq™ HS, which is an enzyme for hot start PCR utilizing Taq antibody, with a newly developed buffer which provides superior specificity, increased amplification efficiency and high aptitude for high-speed real time PCR. Accordingly, a successful real time PCR is promised with high sensitivity, wide dynamic range and accurate quantification. This is supplied with ROX™^*2 Reference Dye to nomalize the fluorescent signal between reactions, for instruments that are equipped with this option.

Applicable real time PCR instruments;

• Thermal Cycler Dice™ Real Time System (TaKaRa Cat.# TP800)
• ABI PRISM^® 7000/7700/7900HT,^*3 Applied Biosystems 7300/7500 Real-Time PCR System,
  7500 Fast Real-Time PCR System (Applied Biosystems)
• LightCycler^® (Roche Diagnostics) ^*4
• Smart Cycler^®(Cepheid)^*5
• Mx3000P^® (Stratagene) ^*6
• Chromo4™ (Bio-Rad Laboratories) ^*7

*1 SYBR^® Green I is provided under licensing agreement with Molecular Probes Inc. for internal research use only.
SYBR^® is a registered trademark of Molecular Probes Inc.
*2 ROX™ is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.
*3 ABI PRISM^® is a registered trademark of Applera Corporation.
*4 LightCycler^® is a registered trademark of Roche Diagnostics.
*5 Smart Cycler^® is a registered trademark of Cepheid.
*6 Mx3000P^® is a registered trademark of Stratagene.
*7 Chromo4™ is a registered trademark of Bio-Rad Laboratories.

Storage

Stable at 4°C for 6 months.
It should be protected from light, and careful attention should be made not to cause contamination.

* This product is shipped on dry ice. After delivered, store at 4°C protected from light. Make the concentration uniform by gently inverting a tube before use.

* The storage at -80°C is most recommended for long term storage. Once thawed, store at 4°C and use up within 6 months.

[Caution]
The enzyme activity may be lowered when stored in a freezer set at -20°C. This is because some freezers do not strictly control the temperature, accordingly SYBR^® Premix Ex Taq™ can be thawed in a freezer. The most secured storage is to keep the products at -80°C before use, and store SYBR^® Premix Ex Taq™ at 4°C after thawed.

Content (250 units)

SYBR® Premix Ex Taq™ (Perfect Real Time) (2×conc) *		1.0 ml×5
ROX™ Reference Dye (50×conc.) **		200 µl
ROX™ Reference Dye II (50×conc.) **		200 µl

* contains TaKaRa Ex Taq™ HS, dNTP Mixture, Mg²⁺, and SYBR^® Green I.
** ROX™ Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities among wells when used with real time PCR instruments that have this option.

Specification of TaKaRa Ex Taq™ HS

Unit definition: One unit is the amount of the enzyme that will incorporate10 nmol of dNTP into acid-insoluble products in 30 minutes at 74°C with activated salmon sperm DNA as the template-primer.

Reaction mixture for unit definition:

25 mM		TAPS (pH9.3 at 25°C)
50 mM		KCl
2 mM		MgCl2
1 mM		2-mercaptoethanol
200 µM		each dATP, dGTP, dTTP
100 µM		[α-32P]-dCTP
0.25 mg/ml		activated salmon sperm DNA

Purity: Nicking activity, endonuclease and exonuclease activity were not detected after the incubation of 0.6 µg of supercoiled pBR322 DNA, 0.6 µg of λ DNA or 0.6 µg of λ -Hind III digest with 10 units of this enzyme for 1 hour at 74°C.

Note

TaKaRa Ex Taq™ HS used in this product is an enzyme for hot start PCR utilizing Taq antibody.
Initial denaturation step prior to PCR should be 95°C for 30 sec. It does not require the activation step of heating at 95°C for 10-15 min prior to the PCR.

Application

For Real time PCR quantification of DNA using SYBR^® Green I

Features

• Quick and accurate detection and quantification of target gene through real time PCR.


• Easy-to-use 2 × premix reagent including SYBR^® Green I:
  Ready to perform real time PCR in the presence of a fluorescent intercalator. Just add PCR primers, template and dH₂O to start the reaction.


• High amplification efficiency and highly sensitive detection:
  This kit utilizes an enzyme for hot start, TaKaRa Ex Taq™ HS. As this enzyme-buffer system is optimized for real time PCR, this product offers highly effcient amplification and high sensitivity.


• Convenient:
  A separate tube of ROX™ Reference dye is supplied.


• Versatility:
  Compatible with Smart Cycler^®, LightCycler^®, ABI PRISM^® 7000/7700/7900HT and 7300/7500 Real Time PCR System, Mx3000P^®, Chromo4™.

PCR products:

As most PCR products amplified with TaKaRa Ex Taq™ R-PCR have one A added at 3′-termini, the obtained PCR product can be directly used for cloning into T-vector. Also it is possible to clone the product in blunt-end vectors after blunting and phosphorylation of the end.

PCR test

Consistent amplification results and SYBR^® Green I detection were observed in real time PCR using λ DNA as a template (amplified fragment: 300 bp) and Smart Cycler^® as a real time PCR instrument. Inhibition of Ex Taq™ polymerase activity by the antibody was also confirmed to be more than 90% after an incubation reaction of 55°C for 10 min.