Applied Biosystems 7900HT cycle conditions

Stage 1:
50°C 2 minutes

Stage 2:
95°C 10 minutes

Stage 3 (37 cycles):
95°C 10 seconds
67°C 20 seconds
72°C 40 seconds (data acquired at 72°C)

Stage 4:
95°C 15 seconds
60°C 15 seconds
95°C 15 seconds

Background Allowance

Plates have been validated using 60ng human gDNA with a background of 1µg mouse gDNA as well as human cDNA generated from an amount of RNA equivalent to 125ng with a background of mouse cDNA generated from an amount of RNA equivalent to 2µg.

Detection Thresholds

Ct Values for human primers validated in gDNA fall in the range of 23-35.
All mouse Ct values are greater than 37 cycles.

Ct values for the cDNA-validated primers fall in the range of 22 - 36.9. cDNA was derived from Stratagene QPCR Human Reference Total RNA (CAT. #750500) and Stratagene QPCR Mouse Reference Total RNA (CAT. #750600).

Customers can use above thermocycling conditions and stop at 37 cycles and they will see human gDNA and cDNA amplification and will not see any amplification of mouse gDNA or cDNA material.