Description:
HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix Kit provides optimal performance and maximum convenience for real-time, quantitative analysis of RNA templates in a single reaction format. The RT-PCR process converts and amplifies single-stranded RNA template yielding double-stranded DNA product. One-Step RT-PCR uses gene specific primers, designed to match RNA/cDNA targets, in a single-tube/plate, one-step reaction. This approach offers tremendous convenience when applied to analysis of single targets from multiple RNA samples. Also, it minimizes the possibility of introducing contaminants into reactions between the RT and PCR steps, since both steps are carried out sequentially without opening the reaction tubes/plates between the steps(1-5).
HotStart-IT SYBR Green One-Step qRT-PCR Master Mix Kit includes M-MLV RT and RNase Inhibitor and a 2X Master Mix containing HotStart-IT Taq DNA Polymerase, MgCl2, Ultrapure nucleotides, and SYBR Green I in an optimized reaction buffer. HotStart-IT SYBR Green qPCR Master Mix uses a novel hot start method developed at USB called primer sequestration (Fig. 1). This novel hot start feature increases the specificity and sensitivity of SYBR-based qRT-PCR reactions by substantially reducing primer-dimer formation. With this method, the HotStart-IT protein binds and sequesters primers at lower temperatures making them unavailable for use by Taq DNA Polymerase. Following reverse transcription and the subsequent heat denaturation step, the primer binding protein is inactivated and the primers are released.
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| Fig. 1. HotStart-IT® method. Top Panel: Non-specific products can be generated at low temperatures which causes PCR reaction failure. Bottom Panel: HotStart-IT binding protein blocks non-specific product formation at low temperatures which results in successful PCR reactions. |
This kit exhibits excellent sensitivity as it can detect fewer than 10 target copies, performs over a broad, linear dynamic range of 6 to 7 orders of magnitude, and is compatible with most real-time PCR instruments (Fig. 2). SYBR Green I Dye is used in this kit to detect any double-stranded DNA that accumulates during the amplification process and also allows melt-curve analyses. No fluorescent probes are required. Individual kit components have been carefully formulated to obtain optimal activity of M-MLV RT, Taq DNA Polymerase, and SYBR Green I to allow highly sensitive and specific detection of RNA transcripts from either total RNA or poly(A)+ mRNA. Separate tubes of the passive reference dyes ROX™ and fluorescein are included for added convenience to allow normalization of well-to-well variations.
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| Fig. 2. Real-time PCR Amplification using HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix Kit (PN 75770). GAPDH Assay using HotStart-IT® SYBR® Green One-Step qRT-PCR Master Mix Kit (PN 75770). Duplicate reactions were performed with human placental total RNA as template using an ABI 7500 Fast instrument. The amount of template ranged from 100 ng to 1 pg in an order of magnitude dilution series. The non-specific dsDNA binding dye, SYBR Green I, was used to detect the 122 bp amplicon and ROX was used as a passive reference dye. The amplification process was linear over six orders of magnitude with a correlation coefficient of 0.99 (see inset). The No Template Control (NTC) reaction generated no measurable fluorescence. |
Tested User Friendly™ Functional Test:
HotStart-IT SYBR Green One-Step qRT-PCR Master Mix Kit is a Tested User Friendly™ product, assuring reliable results. Release specifications for the kit are based on the following functional assay. Real-time qRT-PCR reactions were performed on an ABI 7500 Fast Instrument using primers specific to a 122 bp region of the human GAPDH gene and human total RNA as template. Product specifications require that the correlation coefficient from a linear regression over five orders of magnitude (10 pg to 100 ng) must be greater than or equal to 0.95.
HotStart-IT® SYBR® Green qPCR Master Mix (2X), PN 75762:
This Master Mix is a 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl2, Ultrapure nucleotides, and SYBR Green I in an optimized reaction buffer for use in real-time, quantitative PCR reactions. Magnesium and nucleotide concentrations are at 5mM and 0.4mM, respectively.
Kit Components:
100 reaction kit 500 reaction kit
Reaction Volume = 50 µl Reaction Volume = 50 µl
HotStart-IT® SYBR® Green qPCR 2 x 1.25 ml 1 x 12.5 ml
Master Mix (2X)
25mM MgCl2 1 x 1 ml 5 x 1 ml
ROX™ Passive Reference Dye 1 x 100 μl 1 x 500 μl
Fluorescein Passive Reference Dye 1 x 100 μl 1 x 500 μl
M-MLV RT 1 x 40 μl 1 x 200 μl
RNase Inhibitor (10 units/μl) 1 x 40 μl 1 x 200 μl
RNase-Free Water, DEPC-Treated 3 x 1 ml 1 x 15 ml
Brief Protocols
References:
- Goblet, C., Prost, E., and Whalen, R. G. (1989) Nucleic Acids Res. 17, 2144.
- Sambrook, J. and Russell, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 8.46-8.53.
- Sellner, L. N., Coelen, R. J., and MacKenzie, J. S. (1992) Nucleic Acids Res. 20, 1487-1490.
- Roth, M. J., Tanese, N., and Goff, S. P. (1985) J. Biol. Chem. 260, 9326-9335.
- Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491.
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