Geode H14000 User Manual
18
When operating Opal chips
– please note: The maximum time spent between sample loading of
the first Opal chip to starting the Crystal Digital PCR™ run on the Geode instrument shall not
exceed 20 minutes. It is not recommended to prepare more than 3 Opal chips per Geode run at
a time. Refer to the respective Instructions for Use for the Opal chip for detailed instructions on
Opal
chip
operation
for
Crystal
D
igital
PCR™
available
here:
https://www.stillatechnologies.com/technical-resources/naica-system-prism3/
.
Starting a Crystal Digital PCR™ run on the Geode
a) The Geode
Crystal Digital PCR™ reaction steps
The Crystal Digital PCR™ reaction of the Geode instrument involves 3 steps known as
partitioning (droplet generation), cycling (PCR amplification), and release (pressure decrease
and stabilization of temperature). To design a new Crystal Digital PCR™ program, the user
should select the partition, define the temperatures used during the cycling, and add the release
step.
i.
Partitioning
Partitioning is always the
first step of a Crystal Digital PCR™ program. The temperature should
always be set
at 40°C (Sapphire chips) and 25°C (Opal chips) for optimal Crystal Digital PCR™.
To enable droplet generation, pressure increases from atmospheric pressure (AP) to AP + 1000
mbar (followed by a drop to AP + 950 mbar at the end of the partitioning step) If a modification
of the partitioning temperature is required, please contact Stilla Technologies before proceeding.
For Sapphire chips, the generated droplets have an average volume of ~0.68 nL when using the
naica® PCR MIX and the naica® multiplex PCR MIX.
For Opal chips, the generated droplets have an average volume of ~0.24 nL when using the
naica® PCR MIX and the naica® multiplex PCR MIX.
ii.
Thermal Cycling
During the PCR amplification phase, the pressure is fixed at AP + 950 mbar to ensure optimal
Crystal Digital PCR™ performance. If a modification is required, please contact Stilla
Technologies before proceeding.
Stilla Technologies recommends the use of the following cycling parameters for PCR and RT-
PCR
experiments:
Table 2: Crystal Digital PCR™ template called “FAST-Template Stilla PCR 45 cycles” using
the recommended naica® PCR MIX reagent (EvaGreen® dye) with Sapphire chips.
The total
duration of the default program is approximately 2 hours.
Step
Temperature (°C)
Pressure* (mbar)
Duration**
Partition
40*
AP to +950
12 min
Initial denaturation
95
+950
3min
PCR (45 cycles)
95
+950
10 sec
55-65
+950
15 sec
Release
Down to 25
Down to AP
33 min
*Should not be modified, ** Duration states indicative times, variances may occur.
