|
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 |
|
