DTI Brand > Products > PCR
PCR Range
PCR Master Mix
Blue Colour High Fidelity Dye Added Fast Master Mix
PCR Polymerase Enzyme
Routine Taq Polymerase Enzyme
Hot-Start Enabled Taq Polymerase
Prevent Contamination, Ensure Accuracy
Prevent Contamination, Ensure Accuracy
Hot Start Taq for Reliable Results and Glycerol-Free Versatility
Fast LAMP, Accurate Results
End-Point Thermal Cycler
Thermal Cycler is designed specifically to enhance PCR efficiency and accuracy equipped with a 7” sensitive touchscreen and a friendly graphic user interface.
End-Point Instrument
PCR, Others
UPGRADE YOUR PCR SKILLS
| Application | Standard PCR/General Application | Standard PCR/General Application | Colony PCR | High Yield Genotyping | Genotyping |
| Recommended Product | |||||
| Choose For | Routine Use | Routine Use | Fast, Routine PCR | Streamlined Workflow | Value Choice |
| Properties |
| Amplicon size | < 8 kb | < 8 kb | < 5 kb | <15 kb | < 5 kb |
| 5'–3' Exonuclease Activity | Yes | Yes | Yes | Yes (Weak) | Yes |
| 3'–5' Exonuclease Activity | - | - | Yes | Yes | - |
| T/A Overhangs or Blunt | T/A | T/A | T/A | T/A | T/A |
| Speed | 60 sec/kb | 60 sec/kb | 10 sec/kb | 60 sec/kb | 60 sec/kb |
| Hotstart | - | Yes | Yes | - | - |
| Dye Added | - | - | Yes | Yes | Yes |
| Premix | - | - | Yes | Yes | Yes |
What is PCR?
How does PCR work?
Principle of PCR
1. Denaturation: The double-stranded DNA is heated to a high temperature to separate it into two single strands.
2. Annealing: The temperature is lowered to allow short DNA primers to bind or "anneal" to their complementary sequences on the single-stranded DNA.
3. Extension: The temperature is raised slightly to enable the DNA polymerase enzyme to synthesize a new strand of DNA by adding nucleotides to the annealed primers.
Bioinformatics tools plays a crucial role in obtaining and analyzing target gene sequence data.
Commonly Used Bioinformatics Tools:
NCBI Tools: BLAST, Entrez, PubMed, BLAST2GO
Standalone Tools: EMBOSS, Biopython, ClustalW, MEGA
Web-based Platforms: UCSC Genome Browser, Ensembl, Galaxy
How to Retrieve Gene Sequence from NCBI:
• Go to NCBI (ncbi.nlm.nih.gov).
• Choose "Nucleotide" database.
• Search for gene name or symbol.
• Select the correct gene from results.
• Find the sequence on the gene page.
Design and synthesis of amplification primers for the target gene
Primer design guidelines
(commonly calculated using the nearest neighbor method. Approximate formula: Tm = 2(A+T) + 4(G+C) + 35 - 2N.
In the free software Primer3, set 60℃<Tm<65℃)
Types of PCR Enzymes
| Pol I |
| Alpha type |
| Mixed |
| Bacterial DNA polymerase Although it has lower fidelity than the α type, it has a wider range of PCR suitability. Used in general PCR |
| A DNA polymerase derived from archaea. It has 3'→5' exonuclease activity, so it has high fidelity synthesis and is used in PCR, where accurate amplification is required. |
| Pol I and α types mixed in appropriate ratio |
Differences between Pol I and α PCR polymerases
| | Pol Ⅰ (family A) | α (family B) |
| Source organism | Thermophile bacteria | Hyperthermophilic Archaebacterium |
| 3'-5' exonuclease | × | ○ |
| 5'-3' exonuclease | ○ | × |
| 3' end of PCR product | + dA | Blunt |
| PCR enzyme | ・Taq ・EmeraldAmp, etc. | ・PrimeSTAR ・Ex Premier, etc. |
| Example of use | Standard PCR | High fidelity, Long PCR |
Primer design is the most important factor in determining the success or failure of PCR reactions. There are two major considerations for primer design: specificity and efficiency.
• Specifically is determind by the frequency of mispriming events. Primers with poor specificity tend to produce undesired amplicons.
• Efficiency is defined as the ability of primers to amplify a product with a two-fold increase per cycle to the theoretical optimum.
The following tables provide guidelines for primer design.
Guidelines
The optimal length of primers is about 24 or 25 bases. However, length can be between 21 and 28 bases if the melting temperature (Tm) needs to be adjusted. When amplifying long DNA fragments (≥10 kb), 25- to 35-mer primers may provide better results.
The GC content (the number of Gs and Cs in the primer as a proportion of the total number of bases) should be 40–60%.
Having four G and/or C bases at the 3' end might be useful if the primer length is short (the bases provide "a clamping effect"), especially for universal primers, which are typically used for amplifying all cDNA or gDNA in a sample. However, adding these bases may increase non-specific priming events for gene-specific primers.
If possible, design primers with a melting temperature of 68–70°C. While this is not absolutely necessary, using stringent PCR conditions (e.g., "touchdown PCR" and "two-step PCR") can enhance primer specificity.
Primers should be specific to your gene of interest. A BLAST search can be used to find regions of homology. Note: PCR yield often depends on the 3' hexamer of the PCR primer. Primers that form a strong stable duplex actually reduce the amplification efficiency.
Avoid
The maximum number of di-nucleotide repeats in a primer is four (e.g., ATATATAT).
Avoid long runs of a single base (more than three) as this can cause primer slippage and contribute to mispriming.
Forward and reverse primers should not anneal to each other and so should not have complementary G or C stretches (>4 contiguous bases).
Self-complementary 3' ends
Self-complementarity (e.g., within the forward primer) can lead to hairpin formation. A hairpin structure can form with just four G/C base pairs in the stem and three bases in the loop.
The primer melting temperature (Tm) is the estimate of DNA-DNA hybrid stability. Knowing the Tm is critical for determining an appropriate annealing temperature (Ta). A Ta that is too high will result in insufficient primer-template hybridization, leading to low PCR product yield. A Ta that is too low may lead to non-specific product amplification.
Calculation of the Tm of primers shorter than 20 bases can be performed using the Wallace rule:
Tm = 2°C (A+T) + 4°C (G+C)
For accurate estimation of the Tm of primers longer than 20 bases, we recommend using free primer design software such as Primer3.
The final concentration of each primer should be between 0.1 and 0.5 µM. A stock solution of each primer is typically 10–20 µM.
Primer concentrations that are too high increase the chance of mispriming, which may result in nonspecific amplification. Primer concentrations that are limiting can result in extremely inefficient amplification.
Standard desalted primers are satisfactory for most PCR applications.
Should I use a three-step or a two-step PCR protocol?
Three-step PCR includes denaturation, annealing, and extension steps. This type of protocol should be used when the Tm of the primers is lower than the extension temperature or is less than 68°C.
If the melting temperature of the primer (Tm) is close to the extension temperature (72°C) or a few degrees lower, consider using a two-step PCR protocol that includes a denaturation step and a combined annealing/extension step. With this protocol, the annealing temperature should not exceed the extension temperature.
A 68°C extension temperature is preferred for two-step PCR and when amplifying longer templates (>4 kb). This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification.
72°C should be used as the extension temperature when performing three-step standard PCR and for amplification of short fragments (<4 kb).
PCR conditions:
• Use higher denaturation temperatures (e.g., 98°C as opposed to 94°C or 95°C) to allow complete denaturation of the template.
• Keep annealing times for GC-rich templates as short as possible.
• Use primers with a higher Tm (>68°C), because annealing can occur at a higher temperature.
DNA Isolation >
Enzyme Selection >
Thermal Cycling >
Amplicon Analysis >
DNA Isolation
High-quality starting materials are important for the beginning of all good science. We currently offer DNA extraction kits from our in-house India manufactured brand "DTI" as well as from Macherey-Nagel brand which is our global partner of Takara Bio for nucleic acid extraction in the bioanalysis range.
Common nucleic acid isolation methods:
Spin Column
Magnetic Beads
Benefits
• High yield and purity
• Throughput flexibility
• Increased target capture efficiency
• Rapid collection and concentration of sample
Choose our DTI Brand for India manufactured range
DTI, is the premier in-house brand of DSS Takara Bio India Pvt Ltd. specializing in to manufacture routine laboratory molecular biology products within our state-of-the-art facility, which is ISO 9001 and ISO 13485 approved. This certification ensures that our processes and products meet the highest quality standards, enabling researchers, scientists, and professionals to conduct their work with confidence and precision.
Our brand DTI is committed to the visionary initiatives of the Indian government, including Make in India and Atmanirbhar Bharat providing ease of product customization at affordable pricing with reduced lead time.
Product offerings
Choose MN for your DNA application
MN has been a reliable partner for DNA purification for years. Due to the excellent experience in nucleic acid purification, the MN research and technical support team knows all about pitfalls during DNA purification. Therefore, MN developed a variety of optimized solutions for processing your sample material of choice.
Product offerings
PCR Enzyme Selection
The successful amplification of DNA is key to many technologies, whether you're performing next-generation sequencing, cloning, or genotyping. Our large and diverse portfolio of PCR polymerases was designed for success across a variety of applications, from basic and translational research to routine laboratory testing. We create polymerases that simply work faster and better, so you can focus on obtaining the results you need.
PCR Product Offerings
For Routine PCR/Genotyping
For Convenient use dye plus master mix
For High fidelity PCR
For High yield PCR
For Long fragment PCR
For Glycerol-Free options
| > TaKaRa Taq™ HS (Glycerol Free) |
For Lyophilised options
Accessories
Thermal Cycling
DTI FabSpeed Thermal Cycler is designed specifically to enhance PCR efficiency and accuracy. It is equipped with a 7” sensitive touchscreen and a friendly graphic user interface, which makes operation highly intuitive. With flexible ramp rate and gradient temperature control, DTI FabSpeed greatly increases PCR accurate optimization.
Features & Benefits
Flexible Ramp Rate Control | Fully Adjustable Lid Temperature | Gradient Optimization |
| From 0.1 - 5.5 ℃/sec to meet the need for the CRISPR related assays. | Can be set between 35 and 120 °C for virtually any type of experiment including NGS pre-treatment. | Range from 1 to 30 °C enables optimal experimental conditions in a single PCR run. |
Ordering Information
Brand | Catalogue Number | Description |
| DTI Brand | TCST-9622 |
Amplicon Analysis
Agarose gel electrophoresis is a widely used method in PCR workflows for analyzing nucleic acids. It helps separate, identify, quantify, and purify PCR amplicons. Selecting the appropriate products for nucleic acid electrophoresis can greatly enhance and expedite your results.
Our high-quality agarose and markers are designed to support accurate analysis, ensuring you achieve precise and reliable outcomes in your downstream applications.
PCR Product Offerings
| > DL 2,000 DNA Marker |
| > DL 5,000 DNA Marker |
Takara Bio companies provide kits, reagents, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, DSS Takara Bio India Private Ltd. is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.
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