BioMaster RT-PCR – Color (2×)

Application
  • Gene expression analysis;
  • One-step conventional RT-PCR.

Basic description

BioMaster RT-PCR – Color (2×) includes 2× buffer for RT-PCR-Color containing all the necessary components (except for DNA template and primers), BioMaster-mix and DEPC-treated water. The kit is designed for one-step reverse transcription and polymerase chain reaction (RT-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.

The buffer is optimized for both efficient RT and PCR. High density of the solution and marker dyes facilitate gel loading.



Cat. #
2× buffer for RT-PCR-Color BioMaster-mix  
DMSO
DEPC-treated water Number of reactions (50 μl each)
RMС02-40 2 × 0.5 ml 1 × 80 µl 0.5 ml 2 × 0.5 ml 40
RMС02-200 4 × 1.25 ml 2 × 200 µl 0.5 ml 3 × 1.8 ml 200

Product composition 2× buffer for RT-PCR-Color 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, marker dyes.

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.

Limits of use

  • Not recommended for amplicons of ˃ 5 kbp.

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;
  • Allows long-term storage (storage of BioMaster RT-PCR – Color (2×) at room temperature for 2 days and/or multiple thawing-freezing cycles do not affect RT-PCR efficacy);
  • The mix contains dyes that do not interfere with polymerase performance and components increasing sample density for easy gel loading.
 
Note:  Mobility of dyes in 0.5 – 1.5% agarose gel:

Xylene cyanol Bromophenol blue Orange G Tartrazine
10000 – 4000 bp 500-400 bp <100 bp <20 bp

 

Benefits of using

  • High specificity
  • High sensitivity
  • 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)
  • PCR products can be further subjected to TA cloning due to deoxyadenosine overhangs at the ends of amplified DNA fragments.
  • Shorter step of sample preparation for the analysis of PCR results. No loading buffer is required due to the high density of the mixture.

Storage

9 months (under proper storage and transportation conditions).

Transportation

at -20 °С; not more than 30 thawing-freezing cycles.

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