Technical data

Indexing and Sample Processing for Multiplexed Sequencing 5
Step 1B. Amplify the captured libraries with indexing primers containing 6-bp indexes 1–16
SureSelect
XT
Target Enrichment System for Illumina Multiplexed Sequencing 75
2 Prepare t
he appropriate volume of PCR reaction mix, as described in
Table 42, on ice. Mix well on a vortex mixer.
Table 42 Preparation of post-capture PCR Reaction mix
3 Add 35 µL of the PCR reaction mix prepared in Table 42 to each
sample well of a fresh PCR plate or strip tube.
4 Add 1 µL of the appropriate SureSelect PCR Primer Index 1 through
Index 16 (provided in clear-capped tubes). Add only one of the 16
possible indexing primers to each reaction well.
5 Add the DNA library samples to the PCR reactions:
a Obtain the PCR plate or strip tube containing 30 µL of bead-bound
target-enriched DNA samples from ice (prepared on page 68).
b Pipette each DNA sample up and down until the bead suspension is
homogeneous, then transfer 14 µL of the sample to the appropriate
well of the PCR plate or strip tube containing PCR reaction mix and
indexing primer.
c Mix the PCR reactions well by pipetting.
d Store the remaining library-bound beads at –20°C for future use, if
needed.
Reagent Volume for 1
reaction
Volume for 16 reactions
(includes excess)
Nuclease-free water 22.5 µL 382.5 µL
5× Herculase II Reaction Buffer (clear cap) 10 µL 170 µL
Herculase II Fusion DNA Polymerase (red cap) 1 µL 17 µL
100 mM dNTP Mix (green cap) 0.5 µL 8.5 µL
SureSelect ILM Indexing Post-Capture Forward
PCR Primer (orange cap)
1 µL 17 µL
Total 35 µL 595 µL