- PCR-grade Water
- Cell Lysis Enzyme
- Cell Lysis Buffer
- WGA Polymerase
- WGA PCR Buffer
LIBRARY PREP REAGENTS
- Fragmentation Buffer
- Fragmentation Enzyme
- Ligase Buffer
- Ligase Enzyme
- Library Amplification Mix
- Library Amplification Primer
- Nuclease-free Water
- Resuspension Buffer
- 1 x 96 reaction format
- Purification Beads
Required materials not provided
- Laminar flow cabinet
- Pipettes (2, 10, 20, 100, 200, 1000 µl)
- Thermocycler (with hotlid & programmable ramp rate to 25°C/sec)
- Pipette tips (low binding, barrier filter)
- PCR thin walled reaction tube with flat cap (0.5mL or 0.2mL)
- Molecular grade tubes (1.5mL)
- 96 well PCR plate, to suit thermocycler
- Adhesive 96 well plate seal
- 96 well plate centrifuge
- Magnetic stand -96
- 80% Ethanol
- LabChip® GXII Touch™ Nucleic Acid Analyzer and associated reagent kit or Qubit® Fluorometer and associated reagent kit (recommended: Qubit™ 1X dsDNA HS Assay Kit)
- Sodium hydroxide 1N
- Illumina® Sequencer (MiSeq® Instrument or MiniSeq® Instrument)
- Illumina® Sequencer related consumables
- Illumina® Sequencer reagent kit
- MiSeq® Reagent Kit v3 (150 cycle, cat # MS-102-3001)
- MiSeq® Reagent Micro Kit v2 (300 cycle, cat # MS-103-1002)
- MiniSeq® High Output Reagent Kit (75 cycles, Cat # FC-420-1001)
Optional materials not provided
- Multi-channel pipette
- Agarose gel-electrophoresis apparatus
- Electrophoresis power supply
- UV transilluminator or gel documentation instrument
PG-Seq™ 2.0 Manual Version – 4320-0020
PG-Seq™ 2.0 Automation pre-plated for Sciclone – 4323-0030
NGS SUPPORT INFORMATION
Publications that Cite Using the PG-Seq Kit 2.0 for Preimplantation Genetic Testing
Mai, A. D., Harton, G. L., Quang, V. N., Van, H. N., Thi, N. H., Thuy, N. P., . . . Quoc, Q. T. (2020). Development and clinical application of a preimplantation genetic testing for monogenic disease (PGT-M) for beta thalassemia in Vietnam. Journal of Assisted Reproduction and Genetics. doi:10.1007/s10815-020-02006-y.
What does the PG-Seq™ kit 2.0 include?
The PG-Seq™ kit 2.0 includes WGA reagents, library preparation reagents including 96 unique barcodes and purification beads and PG-Find™ software.
How many reactions do I get in a single PG-Seq™ kit 2.0
What is the maximum throughput for the PG-Seq™ kit 2.0?
Does the PG-Seq™ kit 2.0 include primers for the TSE protocol?
No, customers are required to supply their own primer sets.
Can the PG-Seq™ kit 2.0 amplify day 3 biopsies and polar bodies?
How long does it take to process from WGA to sequencer (lab time) for 24, 48 and 96 samples?
24 samples: ~7 hours | 48 samples: ~7.5 hours | 96 samples: ~9.5 hours
How long does the sequencing time take for the v3 and v2 chemistry Illumina® MiSeq® reagent kits?
V3 1×75 cycle: ~ 9 hours | V2 1×75 cycle: ~5 hours
Does the sequencer reagent kit come with the PG-Seq™ kit 2.0?
No, this needs to be purchased from Illumina® or an Illumina® distributor.
What reagent kits do I need for the sequencer?
Illumina® MiSeq® v3 150 cycle kit (MS-102-3001) | Illumina® MiSeq® v2 Micro 300 cycle kit (MS-103-1002)
Illumina® MiniSeq® High output 75 cycle kit (FC-420-1001)
Can I use the PG-Find™ software to view point mutations and SNPs?
No, PerkinElmer recommends using IGV software to view point mutations and SNPs.
What equipment do I need for the PG-Seq™ kit 2.0?
Standard molecular biology equipment and a thermocycler with a programmable ramp rate (please also refer to the kit manual for more information).
At what temperature do I store the kit components?
WGA reagents should be stored at -20°C in the clean room. Library preparation reagents (including the adaptor plate) should be stored at -20°C in the general lab with purification beads stored at 4°C in the general lab. Resuspension buffer & H2O stored at room temperature.
What buffer should I use to transfer the biopsy in and what size tubes shall I use?
Recommended cell transfer buffers include 10 mM Tris-HCl (pH 8.0) (no EDTA) and PBS (Mg2+, Ca2+ free and BSA free) in 0.2 ml or 0.5 ml PCR tubes.
kit 2.0 comes with WGA and library preparation reagents in a 96-reaction kit and the PG-Find™
The PG-Seq™ kit 2.0 also comes in an automation version with the library preparation reagents pre-plated ready to use on the Sciclone® G3 NGSx workstation.
The shelf life of all reagents is 6 months when stored properly at -20°C. The PG-Seq™ kit 2.0 ships on dry ice.
PG-Seq™ 2.0 Manual Version – 4320-0020
PG-Seq™ 2.0 Automation pre-plated for Sciclone® G3 NGSx workstation – 4323-0030