1. Procedure
A. Preparation of the RT- PCR master Mix
1
RBCPolymerases
*
Preparing a master mix as follows:
Component
RBC Therm One One-step
Real-time RT-PCR Premix
For SYGB Green And Taqman probe
Volume / reaction
Final conc.
2x RT-PCR Premix 12.5 μl 1X
Forward Primers (10μM)
Reversed Primers (10μM)
RNA sample
H2O
Store at
-200C
12.5 μl
variable
variable
2~10μl
up to 25 μl
1X
0. 6~1.0 μM
0. 6~1.0 μM
2
RBC ThermOne One-step
Real-time RT-PCR Premix
RBC ThermOne One-step
Real-time RT-PCR Premix
Cat.No.RTR011
Cat.No.RTR012
Component: 1ml X 5vials
Concentration: 2x
Component: 1ml X 5vials
Concentration: 2x
Cat.No.RTR0111
Component: 1ml X 1vial
Cat.No.RTR0121
Component: 1ml X 1vial
Concentration: 2x
Concentration: 2x
For SYBR Green
Mix the master mix thoroughly by pipetting up and down.
3
Quickly spin down the residue of top.
4
Put the mixture on ice.
For Taqman probe
B. Performing One Step RT- PCR
1
Program your instrument as follows:
Step
Temperature
Time
Cycle
Reverse transcription
48˚C
30 min
1
activation step
95˚C
10 min
1
Denaturation
94˚C
30 sec
30~40
Annealing
50˚C ~ 68˚C
30 sec
30~40
Data acquisition
Description
The RBC ThermOne One-step Real-Time RT-PCR Premix is a ready-to-use, 2X concentrated premix that contains all the reagents
(except primers and template) needed for running RT-PCR in a single tube. By mixing powerful reverse transcriptase with Hotstart
Taq DNA polymerase provides high specificity and sensitirity with one-step speed and convenience.
Real-time RT-PCR premix minimizes the risk of contamination and increases the sensitivity of RNA amplification.
2
Place the PCR tubes in the thermal cycle and start the RT-PCR program.
Put the mixture on ice.
Reai-time RT-PCR Premix performance test
Content
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2X Reai-time RT-PCR Premix containing :
Hotstart Taq DNA polymerase.
Reverse transcriptases.
dNTP mix including dATP、dCTP、dGTP、dTTP .
Taqman probe real-time PCR Buffer.
5mM MgCl2 .
High sensitivity of cDNA amplification by RT-PCR is confirmed by using 10 copies viral RNA as the
template(amplified fragment: 0.5kb)