Real-Time Pcr - NEC express5800 A2010b User Manual

PrimeScript™ RT Master Mix （Perfect Real Time）
VIII. Real-Time PCR
The protocol for performing real-time PCR using SYBR® Premix Ex Taq II (Tli RNaseH Plus)
(Cat. #RR820A) after performing a reverse-transcription reaction with this kit is shown below.
If real-time PCR is to be performed with TaqMan® probe detection, please use Premix Ex Taq
(Probe qPCR) (Cat. #RR390A).
< Protocol when using Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
＊ Please follow the procedures provided in the manual of the respective instrument.
1. Prepare the PCR mixture shown below.
<Per reaction>
Reagent
SYBR® Premix Ex Taq II (2X)
PCR Forward Primer (Tli RNaseH Plus) (10 μM)
PCR Reverse Primer (10 μM)
ROX Reference Dye (50X) or Dye II (50X)
RT reaction solution (cDNA solution)
dH O (sterile distilled water)
2
Total
＊ 1: A final primer concentration of 0.4 μM is most likely to yield a good result.
＊ 2: The concentration for ROX Reference Dye II (50X) is lower than that for ROX
＊ 3: In a 20 μl reaction volume, it is preferable to use a quantity of cDNA
＊ 4: Prepare in accordance with the recommended volume for each instrument.
6
Nevertheless, if there is an issue with reactivity, try to find an optimal
concentration between 0.2 and 1.0 μM.
Reference Dye (50X).
• Use ROX Reference Dye II (50X) when performing analyses with Applied
Biosystems 7500 Real-Time PCR System or 7500 Fast Real-Time PCR
System.
• Use ROX Reference Dye (50X) when using StepOnePlus™ or 7300 Real-
Time PCR System.
corresponding to 10 pg - 100 ng of total RNA template. In addition, the
reverse-transcription reaction solution volume should correspond to less
than 10% of the PCR reaction solution volume.
Cat. #RR036Q
v201204Da_2
and StepOnePlus™ Real-Time PCR System >
Amount
10 μl
0.8 μl
0.8 μl
＊2
0.4 μl
＊ 3
2 μl
6 μl
＊4
20 μl
URL:http://www.takara-bio.com
Amount Final conc.
25 μl 1X
＊1
2 μl 0.4 μM
＊1
2 μl 0.4 μM
1 μl 1X
4 μl
16 μl
＊4
50 μl