Prepare Primer Validation Reactions - Fluidigm Access Array User Manual

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

Prepare Primer Validation Reactions

Reagents from Other Suppliers
Product Name
FastStart™ High Fidelity PCR System, dNTPack
Agilent DNA 1000 Kit
PCR Certified Water
60 ng/μL genomic DNA (optional)
Prepare Primer Validation Reactions
The primer validation protocol prepares enough reagents to perform 48 primer validation
reactions.
IMPORTANT It is essential to have Access Array Barcode 1 primers before proceeding with
the primer validation. See Table 1 below for the oligo sequences.
Table 1. Access Array Barcode 1 primers for Ion Torrent
Name
A_BC1_CS1 forward primer
P1_CS2 reverse primer
1 Prepare the 5X target-specific primer solution for 48 individual primer pairs as shown in
the table below.
Table 2. 5X target-specific primer solution preparation
Component
50 μM CS1-tagged TS forward primer
50 μM CS2-tagged TS reverse primer
PCR Certified Water (Teknova)
Total
* The final concentration in this table refers to the amount of each listed component in 5 μL of
the final primer solution.
2 Vortex the 5X target-specific primer solution for a minimum of 20 seconds and
centrifuge for 30 seconds.
NOTE The final tagged TS forward and reverse primer concentrations are 250 nM in the
5X primer solution. The final TS forward and reverse primer concentrations in the PCR
reaction are 50 nM.
3 Prepare 2 μM Access Array Barcode 1 primer solution.
18
Sequence (5'-3')
CCATCTCATCCCTGCGTGTCTCCGACTCAGACGAGTG
CGTACACTGACGACATGGTTCTACA
CCTCTCTATGGGCAGTCGGTGATTACGGTA
GCAGAGACTTGGTCT
Volume
(μL)
1
1
198
200
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Manufacturer
Sigma-Aldrich
Agilent
Teknova
Coriell
Final
Concentration* (nM)
250
250
Part Number
04738292001
5067-1504
W3330
NA17317
Total Length
62 bp
45 bp