Tag Archives: epigenetics

I want to add carboxyl-dCTP to the DNA fragments after RE digestion with Klenow. However, I saw that “5-Carboxy-dCTP is unstable in neutral and acidic conditions.” I wonder if 5-Carboxy-dCTP is stable in NEB buffer 2 (pH=7.9), or what is the best condition to do this. -Venson

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Dear Venson,

Thank you for your inquiry about 5-Carboxy-dCTP. This product was functionally tested when it was added to our catalog.

In a single pase pair extension assay, 5-Carboxy-dCTP incorporated less efficiently than dCTP with Klenow using a buffer of pH 7.9. 5-Carboxy-dCTP was also tested in PCR amplification of a ~500 base pair Lambda gDNA in a pH 8.4 buffer. The PCR product formed with 75% substition of dCTP. Our chemists believe that 5-carboxy-dCTP should be stable enough in NEB buffer (pH 7.9) to be used with Klenow DNA polymerase but please remember to store the product at -20°C.

Please let me know if this provides adequate information for your experiments.

Best regards,
Elizabeth

I recently bought carboxyl dCTP and tried with different polymerases however I was unable to amplify my construct. I tried also C, meC, hmC, fC and they worked fine with AccuPrime, but nothing to do with caC. What do you suggest? Any advice on which polymerase I can use? -Robert

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Dear Robert,

Thank you for contacting TriLink. I am not aware of any specific enzyme which is known to incorporate 5-carboxy-dCTP however; since different analogs incorporate with different efficiencies, I recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions. Alternatively, decreasing the elongation temperature, increasing elongation time in PCR cycle, increasing concentration of dGTP and 5-carboxy-dCTP or adjusting the pH may support incorporation. Please keep in mind this modification is sensitive to pH and should be kept in basic conditions. Do not hesitate to contact us if you have any further questions.

Best regards,
Elizabeth