Experimental Example - NEC express5800 A2010b User Manual

PrimeScript™ RT Master Mix （Perfect Real Time）
IX. Experimental Example
Reverse-transcription reaction time and amount of cDNA synthesized.
[ Method ]
1. Reverse-Transcription Reaction
Reagent: PrimeScript RT Master Mix (Perfect Real Time)
Template: Human Placenta total RNA 2 pg - 2 μg and sterilized water
Reaction volume: 20 μl
Reaction conditions: 37℃ for 15, 30, or 60 min. → 85℃ for 5 sec. → 4℃
2. Real Time PCR
Reagent: SYBR® Premix Ex Taq II (Perfect Real Time)
Template: 2 μl of each of the aforementioned reverse-transcription reactions
Reaction volume: 25 μl
Target gene: Human ACTB
Reaction conditions:
Standard protocol for use with Thermal Cycler Dice Real Time System
[ Results ]
Amplification curve
Standard curve
Violet
Red
Yellow
Reactivity was compared for cases where the reaction time was set at 15, 30, or 60 min.
This experiment showed that reactions could proceed with equivalent efficiency over a
wide range of template concentrations regardless of the reaction time.
URL:http://www.takara-bio.com
Time RSq
15 min. 0.999
30 min. 1.000
60 min. 0.999
Cat. #RR036Q
v201204Da_2
Eff Standard Curve
99.0 Y = -3.346 ＊ LOG(X)+34.52
98.9 Y = -3.349 ＊ LOG(X)+34.46
100.9 Y = -3.301 ＊ LOG(X)+34.24
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