Technical data

Indexing and Sample Processing for Multiplexed Sequencing 5
Step 4. Quantify each index-tagged library by QPCR (optional)
SureSelect
XT
Target Enrichment System for Illumina Multiplexed Sequencing 83
Step 4. Quantify each index-tagged library by QPCR
(optional)
You can use the Agilent QPCR NGS Library Quantification Kit (for
Illumina) to accurately determine the concentration of each index-tagged
captured library. Refer to the protocol that is included with the Agilent
QPCR NGS Library Quantification Kit (p/n G4880A) for more details to do
this step.
1 Prepare a standard curve using the quantification standard included in
the kit, according to the instructions provided in the user guide.
2 Dilute each index-tagged captured library such that it falls within the
range of the standard curve.
Typically this corresponds to approximately a 1:1000 to 1:10,000
dilution of the captured DNA.
3 Prepare the QPCR master mix with Illumina adaptor-specific PCR
primers according to instructions provided in the kit.
4 Add an aliquot of the master mix to PCR tubes and add template.
5 On a QPCR system, such as the Mx3005p, run the thermal profile
outlined in the QPCR NGS Library Quantification kit user guide. Use
the SYBR Green instrument setting.
6 Use the standard curve to determine the concentration of each
unknown index-tagged library, in nM.