The Sybr green PCR Master mix offers Real-time PCR with intercalating dye SYBR Green I
Description
Bio-Star SYBR green Master mix Blue (2×) is developed for quantitative real-time PCR with fluorescent dye SYBR Green I. The Mastermix contains all components, except template and primers, for successful PCR.
– The mix is optimized for efficient and reproducible hot-start real-time
PCR of genomic, plasmid and viral DNA samples.
– The Mastermix contains substances that increase half-life and
processivity of DFS-Taq PLUS DNA polymerase by enhancing its
stability during PCR.
– It includes components that influence primer annealing temperature
and characteristics of template melting thus enabling to increase the
specificity of PCR and use templates with complicated structure.
– DFS-Taq Plus DNA polymerase is inactive at room temperature
because of monoclonal antibodies.
– The inert dye allows control when using multi-well plates. Use of the kit
saves time and minimizes contamination risk due to reduced number of
pipetting steps.
Features:
– Enzyme with hot start capability increases reaction specificity and sensitivity, optimized
– DFS-Taq PLUS DNA polymerase activation requires not more than 5 min heating
– High selectivity and reaction yield
– The mix is colored for easy pipetting
– Reduced preparation time
Applications:
– Real-time PCR with intercalating dye SYBR Green I
– Conventional PCR
– High-throughput PCR
– Genotyping
– For detection of bacterial DNA
Advantages:
– The Sybr green mix is colored for easy pipetting
– Reduced preparation time
– Low chance of contamination while preparing PCR solution
– Standardized conditions of the same-type reactions (reduced pipetting error during mixing PCR components from experiment to experiment)
– Minimized efforts
Components of Sybrgreen Master mix:
2X Bio-Star qPCR-Mastermix SYBR Blue contains:
– 100 mM Tris-НCl (рН 8.5 at 25 °С), 100 mM KCl, 0.4 mM each of dNTP,
3 mM MgCl2, 0.06 U/µl DFS-Taq DNA Polymerase, 0.025% Tween 20,
stabilizers and enhancers, SYBR Green I, Blue inert-dye, which does
not interfere the PCR in a real-time Cycler
– Tube of MgCl2 100 mM
– Water Mol.Bio Grade 2 x 1,25 ml
Storage and transportation: at -20 °С; not more than 50 thawing-freezing cycles. shipping with blue ice or at room temperature
Storage terms: up to 24 mounts
Stability tests / Quality control / Comparison
Exodeoxyribonuclease activity: DNA was stable after incubation of 1 µg fragment of phage lambda DNA in the presence of 25 µl of Bio-Star qPCR Mastermix (2×) in 50 µl reaction solution at 37 °C and 70 °C for 4 h.
Test for bacterial DNA contamination
Test for Hot -Start ( we test activity with or without MAB at various temperatures
Exodeoxyribonuclease activity:
DNA was stable after incubation of 1 μg fragment of phage lambda DNA in the presence of 25 μl of BioMaster HS-qPCR (2×) in 50 μl reaction solution at 37 °C and 70 °C for 4 h.
Ribonuclease activity:
Absence of ribonuclease activity was confirmed after incubation of 1 μg of 5’-[P32]-labeled RNA fragment in the presence of 25 μl of BioMaster HS-qPCR (2×) in 50 μl reaction solution at 37 °C for 4 h.
Stability:
One month at room temperature does not reduce PCR efficiency
| Product | Description |
| Y9220 | BIO-Star PCR Master mix (2x) with SybrGreen PCR master mix Blue 100 rcs (2,5 ml) |
Datasheet
Stability Test Report
Material Safety Datasheet
Recommendations for avoiding contamination during PCR
Over 10 million copies of DNA template are processed during PCR. Therefore, it is important to prevent the possibility of contamination with other templates and amplicons that are present in laboratory. Here are general recommendations for reducing the risk of contamination:
– Preparation of DNA samples, preparation of reaction solutions, amplification and analysis of PCR products should be carried out in different territorial areas.
– Prepare reaction solutions in PCR laminar flow cabinet equipped with UV lamp.
– Use new pair of gloves when purifying DNA and preparing mixtures and solutions.
– Use reagents designed specifically for PCR. Use pipette tips with integrated aerosol filter when preparing DNA samples and reaction solutions.
– For verification of the absence of contamination, prepare a mixture sample without DNA template (negative control).
Recommendations for primer selection
For design of primers and probe, we recommend using Oligo software http://www.oligo.net/ and its analogs. For selection of oligonucleotides, follow the basic principles:
Primers
– Primer length:18-22 bp.
– Difference in melting temperatures (Тm) of the two primers shouldn’t exceed 3 °С.
– Tm of primers for TaqMan PCR should be ≥ 60 °С.
– GC composition of primers should be within the range of 40 to 60%.
– Product length: 70 – 150 bp.
– Minimize secondary structures, avoid them, if possible.
– Check your primers using BLAST.
Probes
– Probe length 22-26 bp;
– Melting temperature: 68-70 °С.
– Minimum of the same nucleotides in a row (especially G: not more than 4 in a row).
– Chose DNA strand that has more C nucleotides than G nucleotides in it.
– There should be no G at 5′-end.
– Avoid self-complementarity and formation of dimers between probe and primers.
DNA sample
– Purity and integrity of DNA is extremely important for successful PCR. For isolation of DNA, apply conventional methods that allow further amplification of the sample.
– Avoid using PCR inhibitors (phenol, hemin, etc.) when working with the samples. In case of using gel purification, minimize UV exposure in order to prevent formation of pyrimidine dimers.
– Prepare reaction solution in a clean area, use pipette tips with integrated filter in order to reduce contamination risk.
– Optimum amount of DNA per reaction depends on the type of sample and its purity: phage lambda DNA ~0.1 ng; E.Coli DNA ~10 ng; human DNA ~10 – 50 ng
Characteristics of amplification steps
– Initial DNA denaturation and enzyme activation
– It is very important to achieve complete denaturation of DNA template at the beginning of PCR which provides its efficient use in the first amplification cycle. If GC composition of the template
is 50% or less, initial denaturation at 95 °С for 5 min will be enough.
Denaturation
Standard time of denaturation per cycle for real-time PCR is considered to be 15 – 30 sec at 95 °C.
Primer annealing and elongation
For TaqMan real-time PCR, annealing and elongation stages are usually combined into one step at 58-60 ºС for 60 sec.
Number of cycles
If there is less than 10 copies of DNA template available per reaction, then efficient amplification requires not less than 40 cycles. A total of 25 – 35 cycles is enough for higher amount of template.