HotStart-IT^® SYBR^® Green qPCR Master Mix with UDG (2X)

HotStart-IT^® SYBR^® Green qPCR Master Mix with Uracil-DNA Glycosylase (UDG) uses a novel hot start method developed at USB called primer sequestration. With this method, a protein binds and sequesters primers at lower temperatures making them unavailable for use by Taq DNA Polymerase (Fig. 1). Following the initial denaturation step, the protein is inactivated and the primers are released. HotStart-IT SYBR Green qPCR Master Mix with UDG is supplied as a 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl₂, Ultrapure nucleotides with an optimized dUTP to dTTP ratio, heat-labile UDG, and SYBR Green I for use in real-time quantitative PCR reactions (qPCR). Simply add DNA template, primers, and water and the reactions are ready for cycling. Separate tubes of passive reference dyes ROX (for ABI and Stratagene instruments) and fluorescein (for BioRad instruments) are included for added convenience.

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Since the mix contains dUTP and UDG, carryover contamination prevention can be performed, which is especially important for high-throughput applications⁽¹⁾. A heat-labile version of UDG that can be irreversibly heat-inactivated is used instead of E. coli UDG, which has been shown to exhibit residual activity following PCR reactions⁽²⁾. The SYBR Green I dye detects any double-stranded DNA that accumulates during the amplification process. The hot start feature enhances SYBRbased qPCR reactions by reducing primer-dimer formation which increases specificity and sensitivity. HotStart-IT SYBR Green qPCR Master Mix with UDG has excellent sensitivity as it detects fewer than 10 target copies, performs over a broad, linear dynamic range of 7 to 8 orders of magnitude, and is compatible with a variety of realtime PCR instruments.

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Real-Time PCR Brochure

Convenient
Save time and reduce potential contamination errors by eliminating several pipetting steps. For a 50 μl reaction, simply add 25 μl of master mix to primers, DNA template and PCR-Qualified H₂O. Reactions can be tailored from 20 μl to 100 μl volumes.

Room temperature reaction assembly is possible because of the hot start feature.

Multiple Platform Compatibility
The SYBR Green I dye is compatible with any instrument’s standard SYBR filter (typically, FAM setting) and the separate ROX and Fluorescein Passive Reference Dyes allow normalization of well-to-well variations that may occur independent of the reactions (e.g., pipetting errors, detection system limitations, etc.).

Carry-Over Contamination Prevention
Eliminate at least 10⁵ copies of dUTP-containing contaminating templates. The dUTP in the mix ensures that amplicons contain uracil which can be destroyed prior to subsequent amplification reactions by the enzymatic activity of the Uracil-DNA Glycosylase. After the initial denaturation step, the UDG is inactivated, and only the desired target sequences without dUTP are amplified.

Advantage with Heat-Labile UDG
The UDG used in the USB master mix, unlike E. coli UDG used in most other products, is completely and irreversibly heat-inactivated due to the hightemperature cycling conditions. This maintains the integrity of the PCR products following reactions which is important if they are to be used in subsequent analyses such as gel electrophoresis, cloning, and/or sequencing.

Novel Hot Start Technology
Since the polymerase is not chemically-inactivated, no extensive heat-activation step is necessary which reduces damage to precious DNA samples.

Higher Specificity, Sensitivity and Broad Dynamic Range
The hot start feature minimizes amplification of non-specific products and primer-dimers. The SYBR Green I concentration has been carefully optimized for maximum sensitivity and can be used in melt-curve analyses. PCR products may be amplified with low background and from low-copy
targets with a linear dynamic range of 7 to 8 orders of magnitude (Fig. 2).

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Stable
Repeated freeze-thaw cycles have no observed effect on performance.

Tested User Friendly™ Functional Tests:
Real-time PCR reactions were performed on an ABI 7500 Fast Instrument using primers specific to a 122 bp cloned region of the human GAPDH gene as template. Product specifications require that the correlation coefficient from a linear regression over seven orders of magnitude (10 to 10⁷ template copies) must be greater than or equal to 0.95.

Additionally, carry-over contamination tests of the UDG activity in the mix were performed. Product specifications require removal of greater than or equal to 10⁵ dUTP-containing template copies per reaction.

HotStart-IT^® SYBR^® Green qPCR Master Mix with UDG Formulation (2X):
The mix combines USB HotStart-IT Taq DNA Polymerase, heat-labile UDG, SYBR Green I, MgCl₂, and Ultrapure nucleotides with an optimized dUTP to dTTP ratio in a unique buffer formulation. Magnesium and nucleotide concentrations are 5mM and 0.4mM each, respectively.

Components:
HotStart-IT^® SYBR^® Green qPCR Master Mix with UDG (2X)
  100 reactions 2 x 1.25 ml 
  500 reactions 12.5 ml
25mM MgCl₂
ROX Passive Reference Dye
Fluorescein Passive Reference Dye
Brief Protocol

References:

1.  Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, 125-128.
2. Thornton, C. G., Hartley, J. L., and Rashtchian, A. (1992) BioTechniques 13, 180-184.

Storage:
Shipped on dry ice. Store at -20°C. Mix well prior to use.