NEC express5800 A2010b User Manual page 7

PrimeScript™ RT Master Mix （Perfect Real Time）
2. Initiate the reaction.
The recommended protocol for PCR is the shuttle PCR standard protocol
described below. Try this protocol first and optimize PCR conditions as necessary.
Perform a 3 step PCR when using a primer with low T
PCR is not feasible.
1) Applied Biosystems 7300/7500 Real-Time PCR System, StepOnePlus™
Figure 1. Shuttle PCR standard protocol
2) Applied Biosystems 7500 Fast Real-Time PCR System
Note:
3. After the reaction is complete, check the amplification and melting curves and plot a
standard curve if an assay will be performed.
Please refer to the instruction manual for your real time PCR instrument to read
about analytical methods.
URL:http://www.takara-bio.com
Shuttle PCR standard protocol
Holding Stage
Reps: 1
95℃ 30 sec.
Cycling Stage
Number of Cycles: 40
95℃ 3 sec.
60℃ 30 sec.
Melt Curve Stage
・ TaKaRa Ex Taq HS in this kit is a hot start PCR enzyme with an anti-Taq
antibody 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.
・ Even for the initial denaturation of template before PCR, 95℃ for 30 sec
is generally sufficient.
Cat. #RR036Q
v201204Da_2
value or when a shuttle
m
Stage 1: Initial denaturation
Reps: 1
95℃ 30 sec.
Stage 2: PCR
Reps:40
95℃
60℃ 30 - 34 sec.
Dissociation stage
＊ : With StepOnePlus™,
set to 30 sec.;
with 7300, set to 31 sec.; and
with 7500, set to 34 sec.
5 sec.
＊
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