Fluidigm Access Array, User Manual

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide
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
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Set Up the PCR Reactions in a 384-Well PCR Plate
1 Prepare the primer validation PCR reactions:
a Add 4 μ L of primer validation reaction mix to each of the 48 wells.
b Add 1 μ L of the 5X target-specific primer solution.
The total PCR reaction volume is 5 μ L.
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 Cycle
50 ºC 2 min 1
70 ºC 20 min 1
95 ºC 10 min 1
95 ºC 15 sec
60 ºC 30 sec
72 ºC 1 min
10
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
2
95 ºC 15 sec
60 ºC 30 sec
72º 1 min
8
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
2
95 ºC 15 sec
60 ºC 30 sec
72 ºC 1 min
8
95 ºC 15 sec
80 ºC 30 sec
60 ºC 30 sec
72 ºC 1 min
5