Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 6: Post-PCR Amplicon Purification and Quantitation
Quantitate PCR Products
37
Stored at Room Temperature
•
DNA suspension buffer (10 mM TRIS, pH 8.0, 0.1 mM EDTA) (Teknova, PN T0221)
•
100% Ethanol
•
PCR Certified Water (Teknova, PN W3330)
Quantitate PCR Products
The PCR product library can be quantified using an Agilent 2100 Bioanalyzer with DNA 1000
Chips.
1
Run 1
μ
L of the 48 pooled PCR products from each sample on a Bioanalyzer DNA 1000
Chip following the manufacturer’s instructions.
a
Ensure that amplicon sizes and distribution are within the expected range (±5% for
amplicons in the range of 200-400 bp including tags).
b
Ensure that primer-dimer contamination in the PCR product pool (in the range of
50
–
130 bp) is less than 25%, based on the Bioanalyzer quantification shown in
Appendix A: Electropherogram Examples
.
2
Continue with the purification procedure below for all of the pooled PCR products that
contain less than 25% primer dimers.
Purify Harvested PCR Products
1
Remove AMPure XP beads from refrigerator and warm to room temperature for
30 minutes.
2
Prepare 70% ethanol solution:
a
To a 15 mL tube, add 3 mL of PCR-Certified Water and 7 mL of 100% ethanol.
b
Vortex for 5 seconds.
3
Pool 1
μ
L of each barcoded sample PCR product (see
Attach Sequence Tags and Sample
Barcodes on page 33
) into a new microcentrifuge tube.
4
Vortex AMPure XP beads for 10 seconds to resuspend. Bead solution should appear
homogeneous and consistent in color.
5
Pipet the barcoded sample pool, DNA suspension buffer, and AMPure XP beads into a
1.5 mL microtube according to the table below:
Component
Volume (
μ
L)
Barcoded sample pool
12.0
DNA suspension buffer
24.0
AMPure XP Beads
36.0
Total
72.0
