Transcription of はじめてのリアルタイム PCR - takara-bio.co.jp
1 PCR PCR Page 1 of 12 PCR PCR PCR PCR DNA RNA SNP PCR PCR PCR PCR PCR PCR PCR RT-PCR PCR PCR 1 DNA 2 2 DNA PCR PCR PCR PCR Page 2 of 12 DNA PCR DNA Threshold Ct Threshold Cycle Ct Ct PCR PCR Page 3 of 12 PCR PCR PCR PCR PCR
2 PCR PCR DNA PCR PCR 96 Thermal Cycler Dice Real Time System PCR Smart Cycler System Thermal Cycler Dice Real Time System III Applied Biosystems 7500 PCR (Thermo Fisher Scientific ) LightCycler Smart Cycler (Roche Diagnostics ) (Cephied ) PCR PCR Page 4 of 12 PCR PCR 2 DNA DNA PCR DNA PCR PCR Page 5 of 12 5 - 5 3
3 DNA Taq DNA 5 3 PCR PCR Page 6 of 12 CycleavePCR RNA DNA RNase H RNaseH RNA RNA RNaseH SNPs PCR PCR PCR DNA Tm PCR DNA PCR PCR Page 7 of 12 Perfect Real Time RT-PCR RefSeq Ensembl Plants
4 PCR TB Green RT-PCR PCR Thermal Cycler Dice Real Time System II TP900 PCR RT-PCR PrimeScript RT reagent Kit (Perfect Real Time) R037A TB Green Premix Ex Taq II (Tli RNaseH Plus) RR820A total RNA PrimeScript RT reagent Kit (Perfect Real Time) 1.
5 RNA RNA PCR PCR Page 8 of 12 1 ( ) 5 PrimeScript Buffer (for Real Time) 2 l 1 PrimeScript RT Enzyme Mix I l mM Oligo dT Primer 50 M *1 l 25 pmol Random 6 mers (100 M) *1 l 50 pmol total RNA RNase Free dH2O Total 10 l *2 *1 Random 6 mers Oligo dT Primer mRNA cDNA Gene Specific Primer Oligo dT Primer (50 M) l 25 pmol Random 6 mers 100 M l 50 pmol Gene Specific Primer (2 M) l 1 pmol *2 10 l 500 ng total RNA 2.
6 37 C 15 *3 85 C 5 4 C *3 Gene Specific Primer 42 C 15 PCR 50 C PCR TB Green Premix Ex Taq II (Tli RNaseH Plus) Thermal Cycler Dice Real Time System 1. PCR 1 PCR PCR Page 9 of 12 TB Green Premix Ex Taq II (Tli RNaseH Plus) 2 l 1 PCR Forward Primer 10 M) 1 l M *1 PCR Reverse Primer 10 M 1 l M *1 Template 100 ng 2 l *2 l Total 25 l*3 *1 primer M M *2 template DNA template 100 ng RT-PCR cDNA (RT )
7 Template PCR 10 *3 25 l 2. PCR PCR PCR PCR PCR Tm PCR 3 PCR 95 C 30 PCR 40 95 C 5 60 C 30 PCR PCR RNA PCR PCR Page 10 of 12 PCR PCR 10 PCR mRNA
8 RNA 3 RNA X mRNA X H RNA A B C 3 PCR X H 6 RNA A B C Ct PCR PCR Page 11 of 12 X H Ct *1 Ct *1 1 1 - 1 - 2 10 - 10 - 3 100 - 100 - 4 1000 - 1000 - 5 10000 - 10000 - 6 100000 - 100000 - RNA A - - RNA B - - RNA C - - X H X *1 *1 *2 *3 RNA A RNA B RNA C *1 Ct X *2 RNA X H *3 "1" RNA A RNA A PCR PCR Page 12 of 12
9 X A "1" B C " " " " PCR PCR PCR
