dUTP Mix, 50mM, Lithium Salt
A mix of ready-to-use sequencing grade ultra-pure dNTPs containing 10 mM dATP, dGTP, dCTP and 20 mM dU, supplied as lithium salts in purified water at pH 7.5, enzymatically synthesized from premium quality raw materials, using highly specific production systems in our purpose-built facilities. The manufacturing process eliminates impurities and PCR-specific inhibitors such as modified nucleotides, tetraphosphates and pyrophosphates commonly observed in other commercially available dNTP products (>99% purity determined by quantitative HPLC). Lithium salts have greater resistance to repeated freezing and thawing cycles than sodium salts, and lithium salt dNTP preparations remain sterile over the entire shelf-life due to the bacteriostatic activity of lithium towards various microorganisms.
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dUTP Mix, 50mM, Lithium Salt achieve the highest dNTP quality, with minimal batch-to-batch variations. They are manufactured to the highest purity standards and are perfectly suited for even the most demanding applications, such as next-generation sequencing (NGS), long PCR, or highly sensitive qPCR.
dNTPs or deoxynucleotide triphosphates are the “building blocks” for DNA. Purity and stability of dNTPs are two of the essential factors to achieve a successful PCR. The use of a highly purified dNTP preparation is particularly recommended for sensitive techniques such as long-range PCR, RT-PCR, multiplex PCR, mutagenesis experiments and real-time applications. The purity of dNTPs is also important when the starting amount of template is minimal.
The standard concentration of each dNTP in a PCR reaction is 0.2 mM. If the starting stock is a 100 mM solution of each dNTP, you need to add 0.1 µL of each nucleotide to a 50 µL standard PCR reaction. Since this is not convenient, it is recommended to prepare mixes: If the 100 mM dNTP stock solutions are mixed in equimolar amounts, the concentration of the mix will be 100 mM total or 25 mM of each nucleotide. From a 100x stock, you need to add 0.5 µL to a 50 µL reaction. Meridian also offers more diluted mixes of 40mM total (10mM each), which is a 50x stock solution and 10 mM total (2.5 mM each) 10x working stock.
It depends. You probably can increase the DNA yield but you will have to optimize the complete PCR reaction, adjust the buffer, the Mg2+ and so on. It is not a matter pertaining only to nucleotides.
Yes. The quality of dNTPs is especially important for sophisticated reactions such as amplification of long templates and real-time PCR. Methylated and deaminated nucleotides exhibit inferior results with proofreading DNA polymerases.
Yes. The optimal pH for storage of nucleotides is from pH 7.5-8.2 (pH at 20 °C). An acidic pH will cause hydrolysis of dNTPs to dNDPs and dNMPs, rendering them less suitable for PCR applications. During freezing/thawing cycles, the pH of the dNTP solutions can differ from the pH at 20 °C. The pH for lithium salt solutions is not as temperature-dependent as with sodium salts, hence where lithium salts are used, no dramatic shifts in pH occur when dNTPs are repeatedly frozen and thawed. This results in the dNTP preparation being more stable and, consequently, having a much longer shelf life than with sodium salts.
The enzymatic synthesis of dNTPs uses highly specific enzymatic systems which eliminate impurities and PCR inhibitors, such as modified nucleotides, PPi and deoxynucleoside tetraphosphates. PCR reactions are impeded by the presence of contaminants resulting from chemical manufacturing processes, such as traces of dNDPs, pyrophosphates or other ionic species (e.g. acetate). Such contamination may lead to poor yields or to no PCR product at all. Unless thoroughly purified, chemically synthesized dNTPs often contain deoxynucleoside tetraphosphates which are powerful PCR inhibitors. Chemical synthesis can also lead to deamination and other nucleotide modifications whereas enzymatic synthesis of dNTPs bypasses these risks.
Reference was made earlier to the greater solubility of dNTPs in lithium salts than in sodium salts. Also, dNTPs presented in lithium salts are more resistant to repeated freezing and thawing than those presented in sodium salts. Furthermore, they remain sterile during the entire storage period (the lithium ion has been shown to have significant bacteriostatic activity towards various microorganisms). Finally, using lithium-salt nucleotide preparations reduces salt-induced artifacts and increases the legibility of sequencing gels. Lithium salts are highly suited to PCR sequencing and labeling applications.
All the concentrations of our dNTP Mixes are totaled, e.g. our 100 mM dNTP Mix is made up of 25 mM of each dNTP (dATP, dCTP, dGTP and dTTP).
When dUTP is used in place of or in conjunction with dTTP, the resulting PCR product is a suitable substrate for Uracil DNA Glycosylase (UDGase), which allows the user to completely destroy any contaminating DNA from a previous PCR reaction prior to commencing the current amplification. At Meridian we provide dUTP both as a stand-alone product and as part of a dUTP Mix.
We recommend that you dilute your dNTPs using Molecular Biology or PCR Grade Water.
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