- High quality - >99% purity determined by HPLC, ideal for use in PCR reactions
- Long shelf-life - no need for aliquoting
- Convenient format - readily incorporated by all common DNA polymerases
- Enzyme free - DNase, RNase and Nickase free
- Flexible sizes - custom, bulk and OEM sizes available
Hydroxymethylcytosine is a modified DNA base present at detectable levels in mammalian tissue and is important because of its potential role in regulating gene expression during normal embryonic development and disease pathogenesis. Bioline has developed a novel method for producing highly purified 5-hydroxymethyl-2’-dexycytidine-5’-triphosphate (Hydroxymethyl dCTP).
EPIK™ Hydroxymethyl dCTP (EPIK HMdCTP) can be used in a number of molecular biological applications including PCR, where it is readily incorporated by DNA polymerases. This ability to generate PCR products in which cytosine is uniformly replaced by hydroxymethylated cytosine can be applied to: epigenetic/methylation studies, studies of hydroxymethylated DNA/protein interaction and forensic DNA analysis. Further applications include the production of DNA fragments resistant to cleavage by a wide range of restriction endonucleases and can be useful in the generation of cDNA libraries.
EPIK HMdCTP is supplied as lithium salts in purified water, as lithium salts have greater resistance to repeated freezing and thawing cycles than sodium salts and remain sterile due to the bacteriostatic activity of lithium towards various microorganisms, giving greater reliability and an extended shelf life.
As an ISO 13485 certified primary manufacturer of nucleotides, if your requirements for EPIK HMdCTP is beyond the scope of our standard product, Bioline can also offer custom, bulk and OEM services (firstname.lastname@example.org).
- DNA methylation studies
- Studies of hydroxymethylated DNA/protein interaction
- Labeling of DNA in vitro
- Substitution of dCTP in a wide variety of molecular biology assays
- Structural and activity studies of the restriction/modification systems of different organisms
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Product SelectionPlease refer to the PCR Selection Chart to confirm the recommended product for your PCR application.
Certification of Analysis (COAs)
EPIK Hydroxymethyl dCTP
1 x 250 μL
Storage & Stability
All components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided. For long-term storage, aliquoting is recommended.
When stored under the recommended conditions and handled correctly, full activity of the reagents is retained until the expiry date indicated on the outer box label.
On Dry Ice or Blue Ice.
Your PCR assay can be dramatically affected by a dNTP preparation containing inhibitors, which have resulted from an inadequate manufacturing process. Several parameters must be taken into account when purity is sought and each dNTP should preferably be free of ribonucleoside triphosphates, other dNTPs, modified nucleotides (methylated, deaminated etc.), deoxynucleoside di- and monophosphates (dNDPs and dNMPs), heavy transition metals, inorganic pyrophosphates (PPi) and nucleoside tetraphosphates.
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.
The standard concentration of each dNTP in a PCR reaction is 0.2 mM. If the starting stock is a 100mM 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 100mM total or 25 mM of each nucleotide. From a 100x stock, you need to add 0.5 µL to a 50 µL reaction. Bioline 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. Several different dNTP Mix formats are available from Bioline as ready-to-go solutions.