Fluidigm Access Array - Page 19

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
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Table 3. The 2 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
(μL)
Final Concentration
(M)
100 M A_BC1_CS1 forward primer 2.0 2
100 M P1_CS2 reverse primer 2.0 2
PCR Certified Water (Teknova) 96.0
Total 100
Component Volume
(μL)
Volume
for 60 (μL)
Final Concentration*
* The final concentration in this reaction mix table refers to the amount of each listed component in 5 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 M each
5 U/L FastStart High Fidelity Enzyme Blend
(Roche)
0.05 3.0 0.05 U/L
20X Access Array Loading Reagent
(Fluidigm)
0.25 15.0 1X
2 M Access Array Barcode 1 primers for Ion
Torrent (from step 3)
1.0 60.0 400 nM
60 ng/L genomic DNA
(Coriell)
0.83 49.8 10 ng/L
PCR Certified Water (Teknova) 0.12 7.2
Total 4.0 240.0