BioMaster RT-PCR SYBR Blue (2×) reagent kit includes 2× buffer for RT-PCR with SYBR containing all the necessary components (except for DNA template and primers), intercalating dye SYBR Green I, BioMaster-mix and DEPC-treated water. The kit is designed for one-step reverse transcription and real-time polymerase chain reaction (qRT-PCR).
BioMaster-mix contains the optimal ratio of M-MuLV –RH to HS-Taq DNA polymerase for both reactions.
M-MuLV –RH is a genetically modified Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV). The enzyme exerts RNA- and DNA-dependent polymerase activity but lacks RNase H activity. M-MuLV –RH reverse transcriptase exhibits improved thermal stability and is active at high temperatures (up to 50°С).
HS-Taq DNA polymerase is a recombinant Taq DNA polymerase inactivated by specific monoclonal antibodies. The enzyme is inactive at temperatures up to 70 °C, it is activated at the first PCR cycle during a short 5-min incubation at 95 °C. Recombinant HS-Taq DNA polymerase catalyzes 5’→3’ synthesis of DNA and possesses 5’→3’ exonuclease activity of the native Taq DNA polymerase from Thermus aquaticus. Recombinant HS-Taq DNA polymerase is ideal for conventional PCR of the templates of up to 5 kbp.
2× buffer for RT-PCR with SYBR is optimized for both efficient RT and real-time PCR. It contains additives and enhancers that enable efficient RT-PCR of complicated and GC-rich templates. Blue hue of the reaction solution provided by the inert dye allows control when using multi-well plates.
Product composition
Cat. #
2× buffer for RT-PCR with SYBR
BioMaster-mix
DMSO
DEPC-treated water
Number of reactions
(50 μl each)
RM04-40
2 × 0.5 ml
1 × 80 µl
0.5 ml
2 × 0.5 ml
40
RM04-200
4 × 1.25 ml
2 × 200 µl
0.5 ml
3 × 1.8 ml
200
2× buffer for RT-PCR with SYBR contains:
100 mM Tris-НCl (рН 8.3 at 25 °С), 150 mM KCl, 0.6 mM each deoxynucleoside triphosphate, 6 mM MgCl2, 8 mM DTT, enzyme stabilizers and enhancers, fluorescent dye SYBR Green I and inert dye.
BioMaster-mix contains:
50 mM Tris-НCl (рН 8.0 at 25 °С), 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 50 % (v/v) glycerol and 0.1 % (v/v) NP-40, M-MuLV –RH reverse transcriptase and HS-Taq DNA polymerase.
SYBR Green I
SYBR Green I is a fluorescent intercalating dye for quantitative and qualitative detection of PCR products during real-time PCR. SYBR Green I provides easy and economical way for detection and quantitative assessment of PCR products during real-time PCR without a need of using specific fluorescent probes. During amplification, SYBR Green I dye penetrates into the minor groove of DNA products and emits stronger fluorescent signal than the unbound dye. Absorption and emission maxima of SYBR Green I are 494 nm and 521 nm, respectively, which enables its usage with any real-time PCR platform existing to date.
Inert dye
The inert dye included in 2× buffer for RT-PCR with SYBR does not reduce PCR efficiency; it facilitates monitoring of multi-well plate pipetting. Absorption maximum of the blue dye is 615 nm.
Limits of use
The presence of the fluorescent dye renders usage of fluorescent probes.
Areas of use
Gene Expression Analysis
RT-PCR one- and two-step
Reaction mixture properties
The reaction mix is optimized for the specific and effective performance of M-MuLV –RH reverse transcriptase and HS-Taq DNA polymerase;
The mix contains substances that allow long-term storage (storage of BioMaster RT-PCR SYBR Blue (2×) at room temperature for 2 days and/or multiple thawing-freezing cycles do not affect RT-PCR efficacy);
The mix contains fluorescent dye SYBR Green I, which allows real-time monitoring of PCR.
Benefits of using
The mix is colored for easy pipetting
High specificity
High sensitivity (1pg – 1 µg of RNA)
Easy and convenient in use
Low pipetting error and low risk of cross-contamination
Standardized conditions of the same-type reactions (reduced pipetting error during mixing PCR components in a series of experiments)
Storage
9 months (under proper storage and transportation conditions).
Transportation
at -20 °С; not more than 30 thawing-freezing cycles.
Product passport
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