Set Up The Pcr Reactions In A 384-Well Pcr Plate; Run The Pcr Reactions - Fluidigm Access Array User Manual

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

Set Up the PCR Reactions in a 384-Well PCR Plate

Set Up the PCR Reactions in a 384-Well PCR Plate
1 Prepare the primer validation PCR reactions:
a Add 4 μL of primer validation reaction mix to each of the 48 wells.
b Add 1 μL of the 5X target-specific primer solution.
The total PCR reaction volume is 5 μL.
2 Vortex the 384-well PCR reaction plate for a minimum of 20 seconds and centrifuge for
30 seconds.

Run the PCR Reactions

1 Load the 384-well plate onto the PCR thermal cycler.
2 Run 35 cycles of PCR using the protocol described below:
Table 5. PCR protocol
PCR Stages
50 ºC 2 min
70 ºC 20 min
95 ºC 10 min
95 ºC 15 sec
60 ºC 30 sec
72 ºC 1 min
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
95 ºC 15 sec
60 ºC 30 sec
72º 1 min
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
95 ºC 15 sec
60 ºC 30 sec
72 ºC 1 min
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
20
Cycle
1
1
1
10
2
8
2
8
5
Access Array System for the Ion Torrent PGM Sequencing System: User Guide