Evaluation of a Homemade SYBR® Green I Reaction Mixture for Real-Time PCR Quantification of Gene Expression (Record no. 46225)

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control field 20250625140701.0
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Classification number (OCLC) (R) ; Classification number, CALL (RLIN) (NR) B-12012
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Title Evaluation of a Homemade SYBR® Green I Reaction Mixture for Real-Time PCR Quantification of Gene Expression
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Volume/sequential designation BioTechniques, 32(4), p.790-796, 2002
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Summary, etc. Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBRR Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScriptÆË reverse transcriptase from 2.5 £gg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the eproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10¡V20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.
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Personal name Karzai A.
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Personal name Üller S.
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Personal name Platz S.
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Personal name Hauser Mt
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Uniform Resource Identifier <a href="https://drive.google.com/file/d/1_nXRmytDDikjpqAhlP2igXiSDxymUuvR/view?usp=drivesdk">https://drive.google.com/file/d/1_nXRmytDDikjpqAhlP2igXiSDxymUuvR/view?usp=drivesdk</a>
Public note Para ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx
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  Clasificación local     Ref1 CICY CICY Documento préstamo interbibliotecario 25.06.2025   B-12012 25.06.2025 25.06.2025 Documentos solicitados