Construction of a standard plasmid for detecting hTERT with real-time PCR

Objective:To construct a recombinant plasmid as the standards for hTERT detection by real-time PCR.Methods:Total RNA was extracted from Hela.A fragment of hTERT was amplified by reverse transcription PCR.The PCR production was ligated with pMD18-T vector and transformed to DH5α.Recombinant plasmid was identified by PCR and sequencing.The concentration of recombinant plasmid was analyzed through its absorption at 260 nm.Standard curve was constructed by real-time PCR with ten-fold serially dilution of recombinant plasmid.Results:A fragment of hTERT was successfully cloned into pMD18-T.Dictated by real-time PCR,standard curve could be received from the concentration of 108 to 102 copys/μl.Conclusion:The standard plasmid for detecting hTERT with real-time PCR has been constructed successfully.