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Leave at room temperature for 2 minutes
Add 1 ul of superscript II RT to each tube. Mix thoroughly and incubate at 25 Cfor 10 min
Incubate tubes a 42Cfor 50 min. Then heat tubes at 70Cfor 15 min. Cool on ice
Add 1 ul RNase H and incubate at 37Cfor 20 min
Store the first strand of cDNA at -20Cfor real-time PCR Real-Time PCR
Normalize the primer concentrations and mix gene-specific forward and reverse primer pair
Every primer concentration should be 5 pmol/ml
Save copy of setup file and delete all PCR cycles
Set up cycles:
1. 50°C 2 min, 1 cycle
2. 95°C 10 min, 1 cycle
3. 95 °C 15 s -> 60 °C 30 s -> 72 °C 30 s, 40 cycles
4. 72°C 10 min, 1 cycl
Prepare mixture in each optical tube (50 ul or 25 ul)
25 ul SYBR green mix (2x)
.5 ul liver cDNA
2 ul primer pair mix (5 pmol/ul each primer)
22.5 ul water