Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC
Prepare Sample Pre-Mix and Samples
29
Prepare Sample Pre-Mix and Samples
All DNA samples need to be added into the sample pre-mix individually, prior to loading the
sample mix solutions into the sample inlets of an IFC.
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 60 reactions. This is enough reagent to
load one IFC with 12 additional reactions to compensate for dead volume and pipetting
error.
Table 8. Sample pre-mix solution
2
Vortex the sample pre-mix for a minimum of 20 seconds and centrifuge for 30 seconds.
Prepare the Sample Mix Solutions
1
Combine the components listed below in a 96-well plate to prepare 48 individual sample
mix solutions.
Table 9. Sample mix solution
Component
Volume
(
μ
L)
Volume
for 60 (
μ
L)
Final
Concentration*
* The final concentration in this sample pre-mix table refers to the amount of each listed component in 5
μ
L of
the final sample 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)
0.1
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
PCR Certified Water (Teknova)
1.95
117.0
Total
4.0
240.0
Component
Volume (
μ
L)
Final Concentration
Sample pre-mix
4.0
genomic DNA (50 ng/
μ
L)
1.0
10 ng/
μ
L
Total
5.0
