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
Prepare Primer Validation Reactions
19
Table 3.
The 2
μ
M
Access Array Barcode 1 primers for Ion Torrent
4
Prepare the primer validation reaction components.
IMPORTANT
Warm the 20X Access Array Loading Reagent to room temperature before
use.
Table 4. 4-primer amplicon tagging primer validation reaction preparation
5
Vortex the primer validation reaction mix for a minimum of 20 seconds and centrifuge for
30 seconds.
NOTE
Table 4 displays the volumes required to prepare 48 reactions with a 25%
overage to evaluate 48 target-specific primer pairs. Scale up appropriately if a higher
number of primer pairs are to be evaluated.
Component
Volume
(
μ
L)
Final Concentration
(
μ
M)
100
μ
M A_BC1_CS1 forward primer
2.0
2
100
μ
M P1_CS2 reverse primer
2.0
2
PCR Certified Water (Teknova)
96.0
Total
100
Component
Volume
(
μ
L)
Volume
for 60 (
μ
L)
Final Concentration*
* The final concentration in this reaction mix table refers to the amount of each listed component in 5
μ
L of the
final reaction mix.
10X FastStart High Fidelity Reaction Buffer
without
MgCl
2
(Roche
®
)
0.5
30.0
1X
25 mM MgCl
2
(Roche)
0.9
54.0
4.5 mM
DMSO (Roche)
0.25
15.0
5%
10 mM PCR Grade Nucleotide Mix (Roche)
0.10
6.0
200
μ
M each
5 U/
μ
L FastStart High Fidelity Enzyme Blend
(Roche)
0.05
3.0
0.05 U/
μ
L
20X Access Array Loading Reagent
(Fluidigm)
0.25
15.0
1X
2
μ
M Access Array Barcode 1 primers for Ion
Torrent (from step 3)
1.0
60.0
400 nM
60 ng/
μ
L genomic DNA
(Coriell)
0.83
49.8
10 ng/
μ
L
PCR Certified Water (Teknova)
0.12
7.2
Total
4.0
240.0
