Features
- Generation of sequencing-ready amplified libraries
- Multiplex clonal amplification: each library pool can include up to 12 uniquely bar coded DNA library samples
- Automated on the GeneRead QIAcube
- Proven performance with the GeneReader NGS System
Product Details
Next-generation sequencing (NGS) is a driving force for numerous applications, including cancer research. After the completion of library preparation using the GeneRead DNA Library Q Kit, the GeneRead Clonal Amp Q Kit is used to prepare sequencing templates using clonal amplification.
Principle
The GeneRead Clonal Amp Q Kit enables processing of DNA library samples and pooling of multiple DNA library samples that have been uniquely bar coded during the library preparation process. The clonal amplification workflow is comprised of emulsion making, emulsion PCR, emulsion breaking and the enrichment of live beads. At the completion of the entire clonal amplification workflow, the DNA library sample/pools will have been clonally amplified and, after enrichment, are ready for sequencing.
Procedure
The 5-step workflow is outlined below:
Library concentration normalization and pooling
- Manual normalization of the DNA library sample(s) concentration and pooling (if multiplexing is to be performed) are performed.
Emulsion making
- The emulsion PCR beads are denatured in preparation for binding to the DNA library pool to eliminate bead clumping.
- Automated emulsion droplet formation is carried out on the GeneRead QIAcube.
- Upon completion of emulsion making, the PCR plate containing the emulsions is removed from the GeneRead QIAcube workdeck and manually loaded onto a stand-alone thermal cycler for clonal amplification.
Breaking and pooling
- The PCR plate containing the clonally amplified emulsions is loaded back onto the GeneRead QIAcube for the automated processes of pooling, breaking and cleaning of the emulsified library pools.
Enrichment
- Super A Beads are manually washed prior to the enrichment of live beads.
- Automated enrichment of live beads is conducted on the GeneRead QIAcube.
- Enriched beads are recovered and manually transferred from the GeneRead QIAcube into sample tubes.
Manual determination of bead concentration using OD
- Following the clonal amplification workflow, a standard curve is created using Primer Loaded PCR Beads that have not been through the clonal amplification process.
- The optical density of the enriched beads is measured so that the concentration can be extrapolated using the standard curve.
- Enriched beads (clonally amplified and enriched library pools) are then ready for sequencing.
Applications
The GeneRead Clonal Amp Q Kit is designed for use in the GeneReader NGS System workflow.
Resources
Safety Data Sheets (1)