The following protocol is intended for real-time detection using the Applied Biosystems 7300 Real-Time PCR System.
The kit also can be coupled with real-time detection methods to conduct realtime quantitative tHDA (qHDA) and RT-HDA (qRT-HDA) to monitor amplification as it progresses. For optimal performance, use EvaGreen as a reporter dye and ROX as a passive reference dye. Sequence-specific probes can also be designed for qHDA experiments.
- Prepare a fresh 7-fold dilution of ThermoScript RT (15 U/μl, Invitrogen, Cat# 12236-022).
- Set up a 50 μl reaction in a MicroAmp optical tube (Applied BioSystems) in a sterile hood or a PCR Workstation.
|10X Annealing buffer II
|MgSO4 (100 mM)*
|NaCl (500 mM)*
|IsoAmp® dNTP Solution
|Forward Primer (5 μM)*
|Reverse Primer (5 μM)*
|IsoAmp® Enzyme Mix
|ThermoScript RT (2.1 U/ìl, Invitrogen)*
|EvaGreen (20X, Biotium)
|ROX Reference Dye (50X, Invitrogen)
- Mix the reaction by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl min eral oil. Place the tubes on ice.
- Real-time detection is carried out on a 7300 Real-Time PCR System (ABI) with the following well inspector setting: reporter dye: SYBR; quencher: none; passive reference dye: ROX. Use the following program:
Stage 1: (60 X)
Step1: 66ºC for 0:05
Step2: 65ºC for 1:55
Data collection and real-time analysis enabled
Stage 2: (1 X)
Dissociation Stage (default settings of the machine)
Melt curve data collection and analysis enabled
* The condition of tHDA reactions can be further optimized by titering thefollowing components:
||Recommended concentration for titering
||3.5 to 4 mM
||3 to 4.5 mM
||30 to 40 mM
||20 to 50 mM
||75 to 100 nM
||50 to 200 nM
||1 to 2 units
||0.5 to 10.5 units