EPIK™ Hydroxymethyl dCTP

Cat No. Size List Price* Qty  
BIO-39046 25µmol
$536.00
 
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EPIK™ Hydroxymethylcytosine (EPIK HMdCTP) is a modified DNA base, and has been specifically developed for Epigenetic applications. Hydroxymethylcytosine also plays a role in regulating embryonic development, pluripotency and stem cell reprogramming. EPIK HMdCTP is supplied as lithium salt and can be used in a number of molecular biological applications including PCR.

Features & Benefits

  • >99% pure by HPLC analysis
  • Readily incorporated by all common DNA polymerases
  • Hydroxymethylated substrates can be ligated by standard ligases
  • DNase, RNase and Nickase free

Applications

  • DNA Methylation studies
  • Studies of Hydroxymethylated DNA/protein interaction
  • Labelling 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

Description

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 has the distinct advantage of being supplied as lithium salt in highly purified water to ensure functionality and long-term stability. Lithium salt has greater resistance to repeated freeze-thaw cycles than sodium salts and lithium salt preparations remain sterile over the entire shelf-life due to the bacteriostatic activity of lithium towards various microorganisms.


Components

Reagent

100mM

EPIK Hydroxymethyl dCTP

1 x 250μl


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.


Shipping conditions

On Dry Ice or Blue Ice.


Certificates of Analysis (COAs)

Certificates of Analysis for EPIK™ Hydroxymethyl dCTP are grouped by catalogue number and then listed by batch number. Larger pack sizes are listed first. To locate the COA for your product, first find the catalogue number and then the required batch number.

View all 5 COAs for EPIK™ Hydroxymethyl dCTP →


Frequently asked questions

  • Why are dNTPs important?

      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.
  • How are Bioline dNTPs supplied?

      dNTPs can be supplied as either a mix or a set. The mix is presented in a single tube containing a premixed solution of dATP, dCTP, dGTP and dTTP. This solution is ready for use and is optimized for PCR and other applications. The set contains four separate tubes, one for each deoxynucleotide. dNTPs can be supplied in lyophilized form or in solution as either lithium or sodium salts.
  • What concentration of dNTPs should I use in PCR?

      The standard concentration of each dNTP in a PCR reaction is 0.2mM. If the starting stock is a 100mM solution of each dNTP, you need to add 0.1µl of each nucleotide to a 50µl standard PCR reaction. Since this is not convenient, it is recommended to prepare mixes: If the 100mM dNTP stock solutions are mixed in equimolar amounts, the concentration of the mix will be 100mM total or 25mM of each nucleotide. From a 100x stock, you need to add 0.5µl to a 50µl reaction. Bioline also offers more diluted mixes of 40mM total (10mM each), which is a 50x stock solution and 10mM total (2.5mM each) 10x working stock. Several different dNTP Mix formats are available from Bioline as ready-to-go solutions.
  • Is it better to use a pre-dispensed dNTP mix rather than a set?

      Yes. A factory pre-dispensed and certified dNTP Mix preparation offers added convenience by minimizing pipetting steps and errors, which could lead to concentration imbalances. And it can be added directly to amplification reactions. Using a dNTP Mix ensures reproducibility in your experiments.
  • Which PCR inhibitors can be present in a dNTP preparation that is not ultra-pure?

      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.
  • Can I increase the DNA yield in my PCR by adding higher concentrations of dNTPs?

      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.
  • Does dNTP quality affect fidelity?

      Yes. Taq polymerase does not discriminate between correct and modified nucleotides, so point mutations may occur. When proofreading DNA polymerases are used, this problem is only partially eliminated since the presence of methylated/deaminated nucleotides often blocks DNA synthesis.
  • Does dNTP quality affect processivity?

      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.
  • Which test is the most stringent quality criterion for dNTPs?

      There is consensus that the most stringent tests to qualify the purity of dNTP preparations are long distance PCR or the synthesis of long cDNAs in a reverse transcription reaction. dNTP quality is also a very important factor for real-time assays.
  • Is the pH of the dNTP solution important for stability?

      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.
  • Is dNTP concentration important for stability?

      Yes. Significant hydrolysis can occur when dNTPs are stored at concentrations below 10mM. When storing nucleotide stock solutions for long periods, ensure that they are at a concentration well in excess of 10mM and preferably at 100mM.
  • Is it important to have a dNTP preparation free from heavy and transition metals?

      Yes. The presence of these metals increases degradation of dNTPs into dNDPs and dNMPs. Hence, metal-free preparations are more stable.
  • What are the advantages of enzymatic synthesis over chemical synthesis of dNTP?

      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.
  • Are nucleotides in solution more stable than the lyophilized version?

      Yes. Preparations of dNTPs decompose into nucleoside di- and mono-phosphates via a disproportionation reaction. At temperatures above 4°C, lyophilized preparations of deoxynucleotides undergo disproportionation faster than nucleotides in solution. By contrast, at -20°C, the rate of degradation for both forms is less than 1% per year. Nucleotides in solution are also generally purer than the lyophilized form. Some lyophilized preparations approach 98% purity or more but rarely match the >99% achieved with extremely pure solutions.
  • What are the advantages of dNTPs being presented in lithium salts as opposed to sodium salts?

      Reference was made earlier to the greater solubility of dNTPs in lithium salts than in sodium salts. Also, as mentioned in the response to question 10, 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 micro-organisms). 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.
  • Is it important to have a dNTP preparation free from inorganic pyrophosphate (PPi)?

      Yes, an excess of inorganic pyrophosphate can inhibit PCR reactions since DNA replication is favored by a low concentration of pyrophosphates on account of the hydrolytic action of cellular pyrophosphates. Inorganic pyrophosphate is often present in chemically synthesized dNTPs and this contamination can be detected by NMR detection and not by conventional HPLC methods. The enzymatic synthesis employed by Bioline produces dNTPs which are entirely free of inorganic pyrophosphate, thus allowing accurate purity determination by HPLC and the identity of the letter and the concentration can be confirmed by photometric measurements.
  • Is it important to have a dNTP preparation free from tetraphosphates?

      Yes, chemical dNTP synthesis is normally carried out by addition of PPi to dNMPs. If PPi is added to dNDP, it will result in the formation of nucleoside tetraphosphates which may cause strand termination as manifested by gel “smearing” and other PCR problems. This is especially important in long-distance PCR applications and reverse transcription of long fragments. Such problems are not encountered with enzymatically synthesized dNTPs, since phosphorylation is carried out using highly specific enzymes that only incorporate one phosphate group at a time.
  • Are the concentrations of your dNTP Mixes each, or total?

      All the concentrations of our dNTP Mixes are totalled, e.g. our 100mM dNTP Mix is made up of 25mM of each dNTP
      ( dATP, dCTP, dGTP and dTTP).
  • I wish to prevent carry-over contamination of my PCR product using dUTP and UDGase, how does this work and which products are suitable?

      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 Bioline we provide dUTP both as a stand-alone product (BIO-39035) and as part of a dUTP Mix (BIO-39041).
  • I need to dilute my dNTPs to a different concentration, what should I use as a diluent?

      We recommend that you dilute your dNTPs using Bioline PCR Grade Water



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