NEC express5800 A2010b User Manual page 8

PrimeScript™ RT Master Mix （Perfect Real Time）
< Method Using Thermal Cycler Dice Real Time System II >
1. Prepare the PCR reaction mixture.
<Per reaction>
Reagent
SYBR® Premix Ex Taq II (Tli RNaseH Plus) (2X)
PCR Forward Primer (10 μM)
PCR Reverse Primer (10 μM)
RT reaction solution (cDNA solution)
dH O (sterilized distilled water)
2
Total
＊ 1: Good results are mostly obtained with a final primer concentration of 0.4 μM,
＊ 2: It is preferable to use a quantity of cDNA corresponding to 10 pg - 100 ng of
2. Start the reaction.
Shuttle PCR standard protocol (below) is recommended. Try this protocol first, and
optimize the reaction condition if needed. When the shuttle protocol is difficult
due to a primer with low Tm value, etc., try a 3 step PCR protocol.
Note:
・ TaKaRa Ex Taq HS is a hot start PCR enzyme with an anti-Taq antibody
・ Even for the initial template denaturation before PCR, 95℃ for 30 sec. is
3. After the reaction is complete, check the amplification and melting curves and plot
a standard curve if a quantitative assay will be performed.
When using Thermal Cycler Dice Real Time System II , please refer to its instruction
manual to read analytical methods.
8
but when there is a problem with reactivity, it is best to consider an optimal
concentration in the range of 0.2 - 1.0 μM.
total RNA template. In addition, the reverse-transcription solution introduced
should correspond to 10% or less of the PCR reaction solution volume.
Figure 2. Shuttle PCR standard protocol
that inhibits polymerase activity. The initial denaturation step prior to PCR
should be at 95℃ for 30 sec. Enzyme activity decreases with longer heat
treatment and the amplification efficiency and quantification accuracy can
be affected.
generally sufficient.
Cat. #RR036Q
v201204Da_2
Amount Final conc.
12.5 μl 1X
1 μl 0.4 μM
1 μl 0.4 μM
＊ 2
2 μl
8.5 μl
25 μl
Hold
(initial denaturation)
Cycle：1
95℃ 30 sec.
2 Step PCR
Cycle：40
95℃ 5 sec.
60℃ 30 - 60 sec.
Dissociation
URL:http://www.takara-bio.com
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