3.5 GEM Incubation

Use a thermal cycler that can accommodate at least 100 μl volume. A volume of 125 μl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers, use highest reaction volume setting.

  1. Incubate in a thermal cycler with the following protocol.

Lid Temperature

 

Reaction Volume

 

Run Time

hh:mm:ss

50°C

100 µl

75 min

Step

Temperature

Time

1

37°C

00:45:00

2

25°C

00:30:00

3

4°C

Hold*

(*proceed to next step immediately after run is at 4°C)

Retrieve Quenching Agent ( PN-2001679) from -20°C and equilibrate to room temperature while the PCR program is running.

*After GEM incubation, proceed immediately to the next step.