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HOME > Protocols > Molecular Biology > Real-time PCR > Protocol for Murine Actin Quantitative RT-PCR (one step)

Protocol for Murine Actin Quantitative RT-PCR (one step)

Primers and Probe

Murine Actin Forward Primer
5'-AGGTCATCACTATTGGCAACGA-3'

Murine Actin Reverse Primer
5'-CACTTCATGATGGAATTGAATGTAGTT-3'

Murine Actin Probe
FAM-TGCCACAGGATTCCATACCCAAGAAGG-TAMRA

Master Mix Recipe (use 5痞 of RNA)

GAPDH Master Mix Components
Volume (無) per Reaction
Concentration in Final Reaction
H2O
15.75無
---
10X TaqMan buffer A
5無
1 X
25 mM MgCl2
11無
5.5 mM
40% glycerol
10無
8%
25 mM dNTPs
1.0無
500然
5 然 murine actin Probe
2無
200nM
5 然 actin Forward Primer
1.5無
150nM
5 然 actin Reverse Primer
1.5無
150nM
M-MLV RT
1.0無
0.25 U/無
RNAse Inhibitor
1.0無
0.4U/無
AmpliTaq Gold
0.25無
0.025U/無

 

Temperature
Time
Cycles
48蚓 30 minutes  
95蚓 10 minutes  
95蚓 15 seconds
40 cycles
60蚓 1.5 minutes

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