Technical data

Indexing and Sample Processing for Multiplexed Sequencing 5
Step 1A. Amplify the captured libraries with indexing primers containing 8-bp indexes A01–H12
SureSelect
XT
Target Enrichment System for Illumina Multiplexed Sequencing 71
Prepare one indexing amplification reaction for each DNA library.
1 Determine the appropriate index assignments for each sample. See
Table 52 in the “Reference” chapter for sequences of the index portion
of the SureSelect 8 bp Indexes A01 through H12 indexing primers used
to amplify the DNA libraries in this step.
Use a different indexing primer for each sample to be sequenced in the
same lane.
2 Prepare the appropriate volume of PCR reaction mix, as described in
Table 38, on ice. Mix well on a vortex mixer.
Table 38 Preparation of post-capture PCR Reaction mix
3 Add 31 µL of the PCR reaction mix prepared in Table 38 to each
sample well of a fresh PCR plate or strip tube.
4 Add 5 µL of the appropriate indexing primer (SureSelect 8 bp Indexes
A01 through H02, provided in white-capped tubes or A01 through H12,
provided in blue plate) to each well. Add only one of the 16 or 96
possible indexing primers to each reaction well.
CAUTION
To avoid cross-contaminating libraries, set up PCR mixes in a dedicated clean area or
PCR hood with UV sterilization and positive air flow.
Reagent Volume for 1
reaction
Volume for 16 reactions
(includes excess)
Nuclease-free water 18.5 µL 314.5 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 31 µL 527 µL