™ ISOFLUX
NGS DNA Kit Catalog No. 910-­‐0104 24-­‐sample kit Instructions for Use Document No. 630-­‐0136 Revision A For Research Use Only Not intended to treat or diagnose any disease condition Fluxion Biosciences, Inc. [email protected] Toll Free US +1 (866) 266-­‐8380 +1 (650) 241-­‐4777 385 Oyster Point Blvd. Suite 3 South San Francisco, CA 94080 IsoFlux™ NGS DNA Kit Instructions for Use INTENDED USE The IsoFlux NGS DNA Kit is intended to be used with the IsoFlux System, a benchtop instrument for semi-­‐automated cell isolations, and one of the IsoFlux enrichment kits. Following enrichment of circulating tumor cells (CTCs) or other rare cells from blood samples, the NGS DNA Kit further enhances the purity of target cells and performs whole genome amplification (WGA), hence generating DNA samples amenable to Next-­‐
Generation Sequencing (NGS). The IsoFlux System and associated products are intended for research applications only. SUMMARY The IsoFlux NGS DNA Kit workflow has two components: purity enhancement and whole genome amplification (WGA). The purity enhancement column and reagents deplete contaminating leukocytes from the magnetic bead pellet following a standard IsoFlux enrichment procedure, enabling high target cell purity that is required by NGS analysis. The WGA reagents are then used to perform amplification of whole genomic DNA (gDNA). The amplified gDNA at a typical concentration of ~100 ng/µL is ready to be used directly in NGS without purification. Document No. 630-­‐0136 Revision A Page 2 of 9 IsoFlux™ NGS DNA Kit Instructions for Use KIT CONTENTS AND STORAGE INFORMATION Box Component Amount Shipping Condition Storage & Stability Purity Enhancement Columns Column Cap Stopper Pedestal 50 mL Tube 1.5 mL Tube 24 pieces 24 pieces 48 pieces 24 pieces 48 pieces 24 pieces Room Temperature Room Temperature Blocking Buffer (10x) 25 mL Ice 4-­‐8 °C. Stable for at least one year. 4-­‐8 °C, protected from light. Stable for at least one year if kept sterile. Dry Ice Store upon receipt at -­‐
70 °C (stable for at least one year). Store at -­‐20 °C after opening (stable for at least 6 months). Purity Enhancement Reagents WGA Reagents Document No. 630-­‐0136 Revision A Release Buffer 11 mL DNA Polymerase (blue lid) 100 µL Reaction Buffer (yellow lid) KOH Buffer (clear lid) Stop Solution (red lid) PBS, 1x (clear lid) DTT, 1 M (lilac lid) 1.5 mL 500 µL 1.8 mL 1.8 mL 1 mL Page 3 of 9 IsoFlux™ NGS DNA Kit Instructions for Use MATERIALS REQUIRED, NOT PROVIDED • IsoFlux Instrument (P/N 950-­‐0100) • One of the IsoFlux enrichment kits (P/N 910-­‐0091, 910-­‐0092, or 910-­‐0103) • Dulbecco's Phosphate-­‐Buffered Saline (DPBS) without Ca2+ and Mg2+ • Tube racks • Tube rotator • Centrifuge • Serological pipettes and pipettor • Calibrated micro-­‐pipettors and tips • Microcentrifuge tubes • Nuclease-­‐free water • Thermocycler WARNINGS AND PRECAUTIONS • For Research Use Only. • Please read the entire contents of these Instructions for Use before processing samples. • All personnel should follow universal precautions for biological sample handling and use laboratory safety equipment (i.e., safety goggles, laboratory coat, and disposable gloves). • All materials coming in contact with the specimen(s) are considered biohazardous. Handle as if capable of transmitting infection. Treat and dispose of waste using proper precautions and in accordance with local, state, and federal regulations. Never pipette by mouth. • KOH Buffer contains potassium hydroxide: Corrosive. Harmful if swallowed. Can cause severe burns. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Document No. 630-­‐0136 Revision A Page 4 of 9 IsoFlux™ NGS DNA Kit Instructions for Use Figure 1. Purity Enhancement Column setup. Each sample requires one Tube A and one Tube B. Tube A houses the column assembled with its cap and stopper. The 50 mL tube itself doubles as a waste collection vessel. After the purity enhancement workflow is completed, the column will be transferred to Tube B for the WGA workflow. Tube B (orange cap) houses an extra clean stopper, a pedestal, and the 1.5 mL DNA collection tube. Document No. 630-­‐0136 Revision A Page 5 of 9 IsoFlux™ NGS DNA Kit Instructions for Use EXPERIMENTAL PROCEDURE Blocking • The purity enhancement columns are shipped pre-­‐assembled (Figure 1). They are designed to stay within Tube A (50 mL waste collection tube) during the purity enhancement workflow. • For each sample, label one column and place it on a tube rack. Remove the cap. • Dilute 1 mL of 10x Blocking Buffer in 9 mL DPBS. Add 9.5 mL into the column reservoir. • Insert the cap securely and incubate at room temperature for ≥ 30 min. • Lift the column, invert, remove the stopper, and invert back into the 50 mL tube. Remove the cap. • Allow the Blocking Buffer to drain completely and then discard the waste. • The column is now ready to receive samples. Purity Enhancement • Take out Release Buffer tubes from the Purity Enhancement Reagents box (4-­‐
8 °C ). Each tube corresponds to one sample. Label the tubes with appropriate sample information, and pre-­‐warm them to 37 °C. • After the IsoFlux enrichment procedure, pipette ~30 µL Binding Buffer into the Low Volume Recovery Holder (provided with 910-­‐0091, 910-­‐0092, or 910-­‐0103), and extract each bead pellet into one pre-­‐warmed tube of Release Buffer. Rinse the Low Volume Recovery Holder at least 3 times with Release Buffer to collect any residual beads. • Screw on the cap tightly and incubate at room temperature on a rotator for 30 min. • Meanwhile, prepare reagents for WGA as described below. • After incubation, slowly pour the Release Buffer into the blocked column, making sure that sample does not overflow. Re-­‐cap the Release Buffer tube and set aside on a tube rack. • Allow the sample to drain completely by gravity flow. • Add 10 mL DPBS into the Release Buffer tube. Invert several times with cap on to rinse the tube. Transfer into the column with a pipette to ensure complete sample extraction. • After the liquid flows through completely, wash the column once more with 10 mL DPBS. • Empty the waste collection tube. • Centrifuge the column for 15s with the maximum speed set at 50x g to remove residual DPBS. Document No. 630-­‐0136 Revision A Page 6 of 9 IsoFlux™ NGS DNA Kit Instructions for Use Whole Genome Amplification • Thaw the DNA Polymerase on ice. Thaw all other reagents at room temperature. Vortex the tubes and centrifuge briefly. The Reaction Buffer may form a precipitate, which will dissolve after vortexing for 10 s. • Reconstitute the KOH Buffer by adding 500 μL nuclease-­‐free water to the tube. Mix thoroughly and centrifuge briefly. The KOH Buffer is pH-­‐labile. Avoid neutralization with CO2. • Prepare the following reagents and keep on ice until use. o Lysis Buffer: Component Volume per Sample DTT 1.5 µL KOH Buffer (reconstituted) 16.5 µL PBS 18 µL o Master Mix (use immediately upon addition of DNA Polymerase): Component Volume per Sample Reaction Buffer 60 µL DNA Polymerase 4 µL Nuclease-­‐free water 14 µL • After the purity enhancement procedure, open Tube B. Remove the clean stopper and insert it into the column. It is important to ensure a snug fit. • Lower a micropipettor into the column reservoir until its tip hovers directly above the membrane. Using visual guidance, carefully pipette 28 µL Lysis Buffer onto the center of the membrane. • Pipette up and down 5 times to facilitate even spreading of the Lysis Buffer across membrane and complete lysis of cells. Take care not to puncture the membrane with the pipette tip. • Incubate the column at 0-­‐4 °C for 10 min. • Add 14 µL Stop Solution. • Add 78 µL Master Mix. Pipette up and down 5 times. The total reaction volume is 120 µL. • Pipette as much as possible of the reaction mix into the 1.5 mL DNA collection tube. • Remove the stopper and secure the DNA collection tube below the column. Place this assembly over the pedestal in Tube B. • Centrifuge at 1000x g for 1 min. • Disassemble the apparatus and transfer the reaction mix into a PCR tube. Document No. 630-­‐0136 Revision A Page 7 of 9 IsoFlux™ NGS DNA Kit •
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Instructions for Use Set up the thermocycler program. Temperature Time 30⁰C 4 hours – overnight 65⁰C 3 min 4⁰C Hold Amplified gDNA can be stored at -­‐80 °C, and used directly in NGS without the need of purification. Minimize freeze-­‐thaw cycles. Concentration determination of amplified gDNA requires quantification methods specific for double-­‐stranded DNA, since the amplification products contain unused reaction primers. Typical concentration is ~100 ng/µL. For PCR analysis, dilute amplified gDNA 1:25 with nuclease free water, and load 2-­‐3 µL in each PCR reaction. Document No. 630-­‐0136 Revision A Page 8 of 9 IsoFlux™ NGS DNA Kit Instructions for Use SUPPORT For assistance with this product, please contact Fluxion Biosciences through one of these methods: Email: [email protected] Phone: Toll Free USA +1 (866) 266-­‐8380 International +1 (650) 241-­‐4777 WWW: www.fluxionbio.com Any product returns should be sent to Fluxion Biosciences ONLY after receiving an RMA number from Technical Support. Fluxion Biosciences Attn: RMA# 385 Oyster Point Blvd. Suite 3 South San Francisco, CA 94080 Document No. 630-­‐0136 Revision A Page 9 of 9