PCR, cDNA amplification
 
 

Master mix for PCR	25µl PCR reaction volume
SYBR® Green PCR Master Mix 2X	12.5µl/tube
H20	10.375 / 9.75 / 8.25µl/tube
Template cDNA	2.0µl/tube (from RT-reaction)

Primers
Add upstream primer (20µM)	0,0625 / 0.375 / 1,125µl/tube (50/300/900nM)
Add downstream primer (20µM)	0,0625 / 0.375 / 1,125µl/tube (50/300/900nM)

PCR-cycling

1. 2 minutes at 50°C for digestion of possible carry over contamination
2. 10 minutes at 95°C for Ampli-Taq Gold activation, followed by a chosen number of cycles (40)
3. 15 seconds at 95°C for melting ( DNA strand dissociation)
4. 60 seconds at 60°C for annealing and extension.
5. Optional: dissociation curve from 60°C to 95°C.

• Prepare the PCR-mastermix for each primer pair (include primers)
• Add template cDNA to tubes separately or to mastermix if same template in many tubes
• Add the PCR-mastermix and template to the reaction tubes
• Spin down reactants
• Perform the PCR