Thermocycler Program

Amplification Protocol

At INVAM, we use either of two PCR machines: the Techne Gradient and the Techne TC312.

Both have lids that will sound an alarm (“beep”) when temperature has been reached and will pause to load samples. Once samples are transferred to heating blocks, each machine varies in steps to begin a preprogrammed PCR protocol.

For the Techne Gradient, the “unpause” button must be pressed twice.

For the Techne TC312, press the [II] button and then press the [enter] button after “to continue” appears on the display screen. Both machines have a similar protocol programmed into them.

Protocol for Amplification of LSU Gene Region

• Heated lid to 105ºC
• Initial denature 94ºC for 4 minutes
• 30 cycles:
• Denature: 94ºC for 30 seconds
• Annealing: 58ºC for 60 seconds
• Elongation: 72ºC for 60 seconds
• Final Elongation: 72ºC for 10 minutes
• Hold: 4ºC