Technical data

5 Post-Capture Sample Processing for Multiplexed Sequencing
Step 1. Amplify the captured libraries
64 SureSelect
XT2
Target Enrichment System for Illumina
Step 1. Amplify the captured libraries
In this step, the SureSelect-enriched indexed library DNA pools are PCR
amplified. The protocol uses half of the bead-bound captured library pool
for amplification. The remainder can be saved at –20°C for future use, if
needed.
Prepare 1 amplification reaction for each captured library pool.
1 Prepare t
he appropriate volume of PCR reaction mixture, according to
Table 27.
Table 27 Preparation of Post-Capture PCR Reaction Mix
Mix well using a vortex mixer and keep on ice.
2 For each amplification reaction, place 35 µL of the PCR reaction
mixture from step 1 in the wells of a SureCycler 8800 PCR plate.
3 Pipette each of the bead-bound captured library pool samples up and
down to ensure that the bead suspension is homogeneous.
4 Add 15 µL of each captured library pool bead suspension to the
appropriate PCR reaction mixture well. Mix thoroughly by pipetting
until the bead suspension is homogeneous. Proceed immediately to
thermal cycling in step 5.
CAUTION
To avoid cross-contaminating libraries, set up PCR master mixes in a dedicated clean
area or PCR hood with UV sterilization and positive air flow.
SureSelect
XT2
Reagent Volume for 1 Amplification
Reaction
Volume for 12 Amplification
Reactions (includes excess)
Nuclease-free water 9 µL 112.5 µL
Herculase II Master Mix 25 µL 312.5 µL
XT2 Primer Mix 1 µL 12.5 µL
Total Volume 35 µL 437.5 µL