DTI dNTP Mix
DTI dNTP Mix
Our dNTPs for PCR are ≥98% pure and are tested for quality control in a variety of applications. Individual dNTPs are supplied at a concentration of 10 mM and can be diluted with water or buffer as needed. The mixture contains each dNTP at a concentration of 10 mM.
SKU:DT0501.1K
DTI dNTP Mix
| Brand | Catalogue Number | Pack Size |
|---|---|---|
| DTI dNTP Mix | DT0501.1K | 1 ml |
Product Description: DTI dNTP Mix (10 mM each) is a mixture of dATP, dCTP, dGTP, and dTTP.
Components: DTI dNTP Mix (10 mM each) - 1 ml
Concentration: 10 mM each
Form: Solution (sodium salt), (pH 7.0 - 9.0)
Storage: -20°C. Avoid repeated freeze-thaw cycles. Once thawed, aliquot into separate tubes and store at –20°C.
Purity: >98%
Performance:
- Good performance was confirmed by PCR amplification of 500 bp and 20 kb fragments from λ DNA template.
- Good performance was confirmed by RT-PCR amplification of a 4.4 kb fragment of the human TFR gene from HL60 cell total RNA.
Applications:
- Ready-to-use: Can be used in PCR reactions without dilution or after diluting to 2.5 mM dNTP.
- RT-PCR
- DNA cloning, sequencing, and labeling.
- Use as a substrate for other DNA amplification and cDNA synthesis reactions.
DTI dNTP Mix - Protocol and Downloads
| Document | Download Link |
|---|---|
| DTI dNTP Mix - Protocol | Download User Manual |
| DTI dNTP Mix - Certificate of Analysis | Download CoA - DT0501.1K |
Experimental Sample Results using dNTP Mix
Consistent amplification of 500 bp fragment from λ DNA is observed using 10 mM dNTP in the RR002M TaKaRa LA Taq® DNA Polymerase (Mg2+ plus buffer) PCR system.
The protocol used for the assay and the results are as follows:
Figure 1: Amplification of 500 bp fragment from λ DNA using dNTP in the PCR system.
Consistent amplification in RT-PCR was confirmed using total RNA from HL60 cells as a template (amplified fragment: TFR region 4.4 kbp). dNTP has been used in the Takara cat# PrimeScript™ RT-PCR Kit RT PCR system.
The protocol used for the assay and the results are as follows:
Figure 2: RT-PCR amplification using dNTP in the PrimeScript™ RT-PCR Kit.
FAQ - DTI PCR Enzymes
What is the role of magnesium in PCR, and what is the optimal concentration?
Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification. Without adequate free Mg2+, PCR polymerases are not active. In contrast, excess free Mg2+ reduces enzyme fidelity and may increase nonspecific amplification. A number of factors can affect the amount of free Mg2+ in a reaction, including DNA template concentration, chelating agents in the sample (e.g., EDTA or citrate), dNTP concentration, and the presence of proteins.
What is the role of salt in PCR reactions?
Successful PCR requires that the DNA duplex separates during the denaturation step and that primers anneal to the denatured DNA. Salt neutralizes the negative charges on the phosphate backbone of DNA, stabilizing double-stranded DNA by offsetting negative charges that would otherwise repel one another. Potassium chloride (KCl) is normally used in PCR amplifications at a final concentration of 50 mM. To improve amplification of DNA fragments, especially fragments between 100 and 1,000 bp, a KCl concentration of 70–100 mM is recommended. For amplification of longer products, a lower salt concentration appears to be more effective, whereas amplification of shorter products occurs optimally with higher salt concentrations. This effect is likely because high salt concentration preferentially permits denaturation of short DNA molecules over long DNA molecules.
It is important to note that a salt concentration above 50 mM can inhibit Taq polymerases.
It is important to note that a salt concentration above 50 mM can inhibit Taq polymerases.
What factors are critical for multiplex PCR?
All primer pairs used in multiplex PCR should have similar priming efficiencies for their target DNA. This can be achieved by using primers with nearly identical optimum annealing temperatures.
When designing primers, pay special attention the following parameters:
When designing primers, pay special attention the following parameters:
- Homology with the target nucleic acid sequence
- Length
- GC content
- Concentration
- Primer homology (primers should not have homology either internally or with one another, especially at the 3' ends)
What is nested PCR?
Nested PCR is a method that involves re-amplification to improve PCR results. Nested PCR involves designing a new forward-nested (FN) or reverse-nested (RN) primer that is internal to the original primer and can pair with the original partner primer. A very small amount of the primary PCR product is used as a template for PCR with nested primers.
Nested PCR frequently leads to improved yield of the desired PCR product by:
Nested PCR frequently leads to improved yield of the desired PCR product by:
- Eliminating extra bands that may have been present in the initial PCR
- Producing a robust band that may have been weak or invisible in the initial PCR
What is touchdown PCR (TD-PCR) and when would I need to use it?
During the PCR denaturation step, all DNA molecules will become single stranded. When the temperature decreases for annealing, three types of duplexes can be formed:
Don, R. H., et al. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008 (1991).
- Homoduplexes—annealing of complementary strands
- Heteroduplexes—cross-hybridization of homologous sequences that may have partial homology
- Duplexes between primers and template
- 5 cycles at 72°C, then
- 5 cycles at 70°C, then
- >25 cycles at 68°C
Don, R. H., et al. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008 (1991).
What is meant by polymerase fidelity? What applications require a high-fidelity polymerase?
The fidelity of a DNA polymerase refers to its ability to accurately replicate a template, or to add the correct nucleotides starting at the 3' end of the primer. The rate of base misincorporation is known as the error rate. PCR polymerases with proofreading activity possess 3'→5' exonuclease activity that can excise incorrectly incorporated nucleotides and replace them with the correct nucleotides.
High-fidelity polymerases are recommended for gene cloning, protein expression, structure-function studies of proteins, cDNA library construction, and next-generation sequencing.
High-fidelity polymerases are recommended for gene cloning, protein expression, structure-function studies of proteins, cDNA library construction, and next-generation sequencing.
Are there any special considerations when handling PCR enzymes?
To avoid contamination, gloves should be worn when handling enzyme tubes, and fingers should be kept away from the tube opening. It is imperative to use a new, clean pipette tip every time you draw from a stock tube of enzyme.
When pipetting enzyme from a stock tube, place the end of the tip just far enough into the liquid to obtain the desired volume. A pipette tip should not be plunged all the way into the enzyme solution as the outside of the tip will become covered with enzyme, preventing accurate measurement and wasting enzyme.
Note: The retention of liquids to polypropylene tips varies with different types of solutions. Pipette tips lose their precision when liquid is drawn more than once. Low-retention pipette tips are recommended for use with viscous solutions, such as those containing glycerol.
When pipetting enzyme from a stock tube, place the end of the tip just far enough into the liquid to obtain the desired volume. A pipette tip should not be plunged all the way into the enzyme solution as the outside of the tip will become covered with enzyme, preventing accurate measurement and wasting enzyme.
Note: The retention of liquids to polypropylene tips varies with different types of solutions. Pipette tips lose their precision when liquid is drawn more than once. Low-retention pipette tips are recommended for use with viscous solutions, such as those containing glycerol.