Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 6)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
Table 5. Thermal cycling protocol.
* Shorter annealing/extension times (1–10 sec) can be used for amplicons <100 bp. Longer annealing/
extension times (30–60 sec or more) can be used for amplicons >250 bp, GC- or AT- rich targets,
low-expressing targets, crude samples, or higher input amounts (for example, 4 µl of cDNA).
Amplification
Real-Time PCR System
Setting/ Scan
Mode
Polymerase Activation and
DNA Denaturation
Denaturation
at 95°C, sec
Annealing/Extension and Plate Read
at 60°C, sec* Cycles Melt Curve Analysis
Bio-Rad ® CFX96 ™ ,
CFX384 ™ , CFX96 Touch ™ ,
CFX96 Touch Deep Well,
CFX384 Touch
™ ,
CFX Connect ™
SYBR ® only
30 sec at
98°C for cDNA
5–15
15–30
35–40
65–98°C
0.5°C increments
at 2–5 sec/step
(or use instrument
default setting)
Bio-Rad ® iQ ™ 5,
MiniOpticon ™ , Chromo4 ™ ,
MyiQ ™
Standard 15–30
Applied Biosystems 7500
and 7900 HT, QuantStudio
Standard 15–30
Roche LightCycler 96 or 480
Fast 10–30
Standard 60
QIAGEN Rotor-Gene and
Stratagene Mx series
Fast 15–30
5. Program the thermal cycling protocol on a real-time PCR instrument according
to Table 5.
6. Load the PCR tubes or plate into the real-time PCR instrument and start the
PCR run.
7. Perform data analysis according to the instrument-specific instructions.
Recommendations for assay design and optimization
■ ■
For best qPCR efficiency, design assays targeting an amplicon size of 70–150 bp
■ ■
The SsoAdvanced ™ Universal SYBR ® Green Supermix and the recommended
qPCR cycling protocols have been optimized for assays with a primer melting
temperature (T
m
) of 60°C designed using the open source Primer3, Primer3Plus,
or Primer-BLAST programs at their default settings. If primers are designed using
other programs, adjust the annealing temperature accordingly