Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC
Attach Sequence Tags and Sample Barcodes
33
Attach Sequence Tags and Sample Barcodes
NOTE All subsequent steps need to be carried out in a post-PCR lab to avoid contamination.
Prepare the Sample Pre-Mix Solution
1 Working in a DNA-free hood, combine the components listed in the table below. This
protocol prepares enough sample pre-mix for 120 reactions. This is enough reagent to
amplify the 48 PCR product pools harvested from one IFC for Set A and Set B
bidirectional barcoding with 24 additional reactions to compensate for dead volume and
pipetting error.
Table 10. Sample pre-mix solution
2 Vortex the sample pre-mix for a minimum of 20 seconds and centrifuge for 30 seconds.
Prepare a 100-Fold Dilution of the Harvested PCR Products
1 In a 96-well plate pipet 99 L PCR Certified Water into 48 wells.
2 Add 1 L of PCR product from each sample harvested from the IFC to a separate well in
the 96-well plate as described in the section Harvest the IFC on page 31.
3 Vortex the PCR product dilutions for a minimum of 20 seconds and centrifuge for
30 seconds.
Prepare the Sample Mix Solutions
1 Combine the components listed below in two 96-well plates to prepare two sets of 48
individual sample mix solutions.
Component Volume
(μL)
Volume
for 120
(μL)
Final
Concentration*
* The final concentration in this sample pre-mix table refers to the amount of each listed component in 5 L of
the final sample mix.
10X FastStart High Fidelity Reaction Buffer
without MgCl
2
(Roche)
2.0 240.0 1X
25 mM MgCl
2
(Roche) 3.6 432.0 4.5 mM
DMSO (Roche) 1.0 120.0 5%
10 mM PCR Grade Nucleotide Mix (Roche) 0.4 48.0 200 M each
5 U/L FastStart High Fidelity Enzyme Blend
(Roche)
0.2 24.0 0.05 U/L
PCR Certified Water (Teknova) 7.8 936.0
Total 15.0 1,800.0
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