Ionic G2 Tissue to DNA Kit

By Bionano Genomics, Inc.

Ionic G2 Tissue to DNA Kit Protocol DOCUMENT NUMBER: CG-00037 DOCUMENT REVISION: BG2 Ionic Tissue to DNA Kit Protocol, Doc. #xxxxx, Rev. A For Research Use Only. Not for use in diagnostic procedures. Kit Contents Table 1: Contents provided in the Ionic® Tissue to DNA Kit Container Item Name Description Volume Quantity per Kit -20⁰C Reagents Box Lysis Buffer 1 Lysis Buffer 1 1.66 mL 6 -20⁰C Reagents Box RNase RNase A Reagent 270 μL 1 -20⁰C Reagents Box Proteinase K Proteinase K Reagent 540 μL 1 -20⁰C Reagents Box Sample Buffer Blank Sample Buffer 1.6 mL 1 RT Reagents Box Lysis Buffer 2 Lysis Buffer 2 3.3 mL 1 RT Reagents Box 1 - Extraction Buffer Extraction Buffer 12 mL 1 RT Reagents Box 2 - Anodic Buffer Anodic Buffer 12 mL 1 RT Reagents Box 3 - Separation Buffer Separation Buffer 18 mL 1 RT Reagents Box 4 - Neutralization Buffer Neutralization Buffer 12 mL 1 RT Reagents Box 5 - Cathodic Buffer Cathodic Buffer 12 mL 1 Fluidic Chip Set Box Ionic Fluidic Chip (Tissue) Fluidic Chips N/A 6 Table 2: Reagents provided by the user. User-supplied Reagents For Lysate Preparation For Purification Fresh/frozen tissue ✓ Table 3: Equipment provided by the user. User-supplied Equipment (or equivalent) For Lysate Preparation For Purification 12-column reservoir (Agilent 204365-100) ✓ P200 multichannel pipette ✓ P200 single channel pipette ✓ ✓ P20 single channel pipette ✓ Microcentrifuge ✓ Programmable ThermoMixer ✓ Vortex mixer (adjustable speed) ✓ ✓ Table 4: Labware provided by the user. User-supplied Labware (or equivalent) For Lysate Preparation For Purification DNA LoBind Tube, 1.5 mL (Eppendorf 22431021) ü ü Optional: DNA LoBind Tube, 2.0 mL (Eppendorf 22431048) ü Optional: DNA LoBind Plate, 96-well (Eppendorf 951032000) ü Protocol at a Glance Lysate Preparation 1. Thaw Lysis Buffer 1 on ThermoMixer at 56°C for 10 minutes at 1400 rpm. 2. Add 90 μL of Proteinase K to a Lysis Buffer 1 tube to make Lysis Mix 1. 3. Collect specimen at bottom of tube by spinning at full speed for 1 minute. 4. Add 195 μL of Lysis Mix 1 to each sample tube. 5. Incubate samples on ThermoMixer: 56°C for 5 hours at 1400 rpm 6. Centrifuge lysate tubes at full speed for 5 minutes. 7. Add 540 μL of Lysis Buffer 2 and 45 μL of RNase into a new tube to make Lysis Mix 2. 8. Add 65 μL of Lysis Mix 2 to eight new tubes. 9. Collect 170 μL lysis reaction from incubated lysate tubes and transfer to tube containing Lysis Mix 2. 10. Incubate at 37°C for 10 minutes. Purification 1. Prepare Purification Buffer Reservoir. 2. Place Ionic Tissue Chip onto instrument. 3. Load Purification Buffers and prime Ionic Tissue Chip. 4. Add 200 μL of each lysate to the chip. 5. Start purification run (~70 minutes). 6. Collect ~50 μL of each extract. Sample Requirements This protocol is intended for the extraction and purification of DNA from up to eight fresh/frozen tissue samples in parallel and is optimized to extract the maximum yield from a minimal amount of tissue. Samples may be minced, pulverized, or OCT-embedded. NOTE: Samples preserved with RNA stabilizers are NOT supported. Bionano recommends an input of 5 mg for most downstream applications. For samples containing small amounts of DNA, up to 10 mg may be used, though care must be taken not to overload the chip. NOTE: Overloading sample can lead to lower yields (see Figure 1). A small pilot study can be helpful in defining optimal input conditions. Some types of tissue samples present challenges to extraction and purification that require optimization beyond the standard protocol. Contact Support@bionano.com for assistance in this case and for advice on processing samples that do not meet the requirements above. Figure 1. Impact of overloading Lysate Preparation – Single Tube Format Preparation before lysis protocol Lysis Buffer 1 1. Completely thaw Lysis Buffer 1 by incubating on the ThermoMixer at 56°C for 10 minutes at 1400 rpm. NOTE: Lysis Buffer 1 must be at room temperature before vortexing and preparing Lysis Mix 1. 2. Add 90 μL of Proteinase K to the tube containing Lysis Buffer 1 to make Lysis Mix 1. 3. Vortex Lysis Mix 1 for 5 seconds and spin down for 10 seconds. NOTE: All centrifuge steps should occur at room temperature. Samples 1. Transfer up to 10 mg of OCT-embedded, minced, or pulverized fresh-frozen tissue to a 1.5 mL or 2.0 mL nuclease-free microtube. NOTE: For OCT-embedded samples, OCT removal is advised, but not required. NOTE: This product is not compatible with tissue specimens that have been stored or treated with high ionic strength RNA stabilizer reagents such as RNAlater. 2. Gather tissue specimen at the bottom of the tube by centrifuging for one minute at full speed (20,000 x g, 14,000 RPM). ThermoMixer 1. Place an appropriate heating block on the ThermoMixer to accommodate sample microtubes. Use of the ThermoMixer with its lid is recommended. 2. Create an incubation program for the Lysis Procedure with the following parameters: Incubate at 56°C for 5 hours at 1400 rpm. Preparation of Lysates 1. Add 195 μL of Lysis Mix 1 to the tissue specimen. 2. Vortex for 5 seconds and centrifuge for 10 seconds. 3. Place sample tubes into ThermoMixer and start the Lysis Procedure program created earlier. NOTE: Align the tube hinge to face the outside of the rotor. The pellet will form on the hinged side of the tube. 4. At the end of the program, remove the lysates from the ThermoMixer and spin them at full speed (20,000 x g, 14,000 rpm) for 5 minutes to pellet debris. 5. Set the ThermoMixer to 37°C, 0 rpm, hold. 6. Label one new 1.5 mL or 2 mL microcentrifuge per lysis reaction. 7. During the spin, add 540 μL of Lysis Buffer 2 and 45 μL RNase to a new tube labeled Lysis Mix 2. 8. Vortex for 5 seconds and briefly centrifuge. 9. Add 65 μL of Lysis Mix 2 to eight labeled tubes (one tube per original sample). 10. After the spin, aliquot 170 μL of the lysis reaction into the tube containing Lysis Mix 2 while avoiding the pelleted debris. NOTE: The pelleted debris may not be visible. Avoid the pellet whether visible or not, by carefully pipetting from the opposite side of the tube from the pellet. If samples are from a fatty tissue such as breast, also avoid the top layer of fat when transferring the lysate. 11. Vortex for 5 seconds and spin down for 10 seconds. 12. Place sample tubes into ThermoMixer set at 37°C and incubate for 10 minutes without shaking. 13. Proceed with Purification or store samples at -20°C for up to 7 days. NOTE: For frozen samples, thaw to room temperature, then vortex and spin briefly before proceeding with the protocol. Purification Prepare Buffer Reservoir 1. Label a 12-channel reservoir as shown in Figure 2 skipping every other column to prevent purification buffer cross-contamination. Figure 2. Reservoir column labels 2. Add purification buffers to each column according to Table 5. Table 5. Reservoir buffers and volumes Reservoir Column Buffer from Kit Volume 1 1 - Extraction Buffer 2.0 mL 2 2 - Anodic Buffer 2.0 mL 3 3 - Separation Buffer 3.0 mL 4 4 - Neutralization Buffer 2.0 mL 5 5 - Cathodic Buffer 2.0 mL Set up Ionic Purification Run 1. From the Ionic Purification System start screen, press Start then select a User profile for the run. 2. Press the G2 Tissue to DNA button. The instrument cover will open. 3. When prompted, remove an Ionic Tissue chip from its packaging, handling only by the side skirting, and place on the instrument stage with the barcode on the right as shown in Figure 3. Gently apply pressure to all four corners of the fluidic chip simultaneously to confirm that the chip is fully seated on the stage. Press the Arrow on the right side of the screen to continue. NOTE: Remove the Ionic Tissue chip from its packaging and place chip directly on the instrument stage to minimize accumulation of static electricity. Avoid contact with the top and bottom surfaces of the fluidics chip. Figure 3. Proper handling and placement orientation of an Ionic Tissue chip. 4. Enter Run Name, Chip ID, and Reagent Lot and press the Arrow on the right side of the screen to continue. NOTES: Chip ID is located on the label on the top surface of the Ionic Tissue chip. Include both the P/N and L/N for the chip: Reagent Lot number is located on the G2 Tissue to DNA Kit room temperature box label. Barcodes can be scanned into the software using a handheld barcode reader connected to the instrument USB port. 5. Using a P200 multichannel pipette and the proper pipetting technique shown in Figure 4, add the appropriate volume of each purification buffer as shown on the screen (also see Figure 5 ), working left to right (1 to 5), to the fluidic chip. NOTE: Do not remove the plastic film from the sample wells at this point as the fluidic chip will not prime correctly without these wells covered Figure 4. Proper (left) and improper (right) pipetting techniques for fluidic chip buffer wells. NOTE: Correct technique is important to ensure the buffers prime correctly into the fluidic chip. Visually inspect tips to ensure each contains the same volume prior to adding to the fluidic chip. Rest pipette tips on the top right of the wells and dispense against the left wall no more than halfway into the well with the pipette at an angle of ~45° relative to the chip surface. Smoothly dispense at a steady speed to the first stop only then remove tips from the well by dragging the tips against the top right wall of the wells. Always avoid contact with the bottom of the wells when dispensing. Change tips after each dispense. Figure 5. G2 Tissue to DNA purification buffer dispense volumes. NOTE: The Separation Buffer should be added using two pipette transfers with fresh tips for each transfer. 6. After loading purification buffers press the Arrow on the right side of the screen to continue. The instrument cover will close, and the fluidic chip will prime. NOTE: Priming takes approximately 4 minutes. NOTE: Samples should be loaded within 10 minutes of priming completion. 7. During fluidic chip priming, vortex lysates for 5 seconds and pulse spin for 10 seconds. Hold on ice. 8. When priming is complete press the Arrow on the right side of the screen to continue. Press OK to confirm samples have been vortexed. The instrument cover will open. 9. Enter sample naming information (See Ionic User Manual for additional information on sample naming options) and press the Arrow on the right side of the screen to continue. Confirm run information is correct and press the Arrow on the right side of the screen to continue. 10. While firmly holding the chip by the side skirt, carefully remove the plastic film from the sample wells using the pull tab and press OK to continue ( Figure 6 ). Figure 6. Remove the plastic film from the sample wells. 11. Using a P200 pipette and keeping the tip in contact with the bottom of the tube, slowly aspirate and transfer 200 μL of lysate to each sample well. NOTE: Insert tip no more than halfway into the well at the position noted in Figure 7 and smoothly dispense at a steady speed to the first stop only. Figure 7. Proper Sample Well lysate loading positioning. 12. Press Begin Run. The instrument cover will close, and the purification process will begin. NOTE: The G2 Tissue to DNA purification run will proceed for approximately 65 minutes and the instrument will indicate when the run has completed. IMPORTANT: Upon completion, extracts should be collected within 1 hour. Collect DNA Extracts 1. Once the purification run has completed, click the Arrow on the right side of the screen to continue. The instrument cover will open. 2. Using a P200 pipette set to 60 μL, aspirate each purified DNA extract (~50 μL) by placing the tip at the bottom of the left wall of each Extraction Buffer well and transfer to a LoBind microcentrifuge tube or LoBind 96-well microplate. NOTE: As demonstrated in Figure 7, the Extraction Buffer well has a small ledge. Be sure to navigate the pipette tip to the left of the small ledge to reach the well bottom. Figure 7. Aspirating the DNA extract from the Extraction Well 3. Using a P20 pipette set to 10 μ