About Hydroxy/Methylated Nucleotides
5-(Hydroxy)methylcytosine dNTP Mix and 5-(Hydroxy)methyl dCTP from Zymo Research are of ultra high purity and can be used to generate DNA by PCR using ZymoTaq™ or other DNA polymerases.
|For 5-hmC||For 5-mC|
|Reagent||Volume||Final Concentration||Volume||Final Concentration|
|2X Reaction Buffer||25 µl||1X||25 µl||1X|
|dNTP Mix||0.5 µl||0.25 mM each dNTP||0.5 µl||0.25 mM each dNTP|
|Forward Primer (10 µM)||Variable||0.3 to 1 µM||Variable||0.3 to 1 µM|
|Reverse Primer (10 µM)||Variable||0.3 to 1 µM||Variable||0.3 to 1 µM|
|Template DNA||Variable||< 200 ng/50 µl||Variable||< 200 ng/50 µl|
|ZymoTaq™ DNA Polymerase (5 U/µl)||1 µl||5 U/50 µl||0.4 µl||<2 U/50 µl|
|ddH20||to 50 µl||-||to 50 µl||-|
|Total Volume||50 µl||50 µl|
|Note: The final concentration of MgCl2 in the reaction (above) is 1.75 mM. If required, scale reaction reagent volumes accordingly to optimize the MgCl2, primer, and/or template concentrations.|
|Concentration||5-Hydroxymethylcytosine dNTP Mix: 2.5 µmol/250 µl as a [10 mM] solution. Contains [2.5 mM] of each dNTP (dATP, dTTP, dGTP and d5hmCTP).
5-Hydroxymethyl dCTP: 10µmol/100 µl as a [100 mM] solution.
5-Methylcytosine dNTP Mix: 2.5 µmol/250 µl as a [10 mM] solution. Contains [2.5 mM] of each dNTP (dATP, dTTP, dGTP and d5mCTP).
5-Methyl dCTP: 1µmol/100 µl as a [10 mM] solution.
|Applications||PCR amplicons containing 5-(hydroxy)methylcytosine are useful in a variety of applications such as:
|Storage||5-(Hydroxy)methylcytosine dNTP Mix and 5-(Hydroxy)methyl dCTP are guaranteed for 12 months at -20°C. Avoid multiple freeze/thaw cycles. For long-term storage, aliquoting is recommended with storage at -80°C.|
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5-hyxroxymethylcytosine-containing DNA substrates were synthesized from a plasmid by utilizing the 5-hmC dNTP mix during PCR amplification. Further analysis utilizing these DNA substrates helped confirm the 5-hmC dehydroxymethylation activities of DNA methyltransferases Dnmt3a and Dnmt3b.
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