DNA Extraction Kit. Instructions For Use (IFU) English

Transcription

1 DNA Extraction Kit Instructions For Use (IFU) English

2 REF Store at C 96 DiaSorin Ireland Ltd. Unit 13/14 Holly Avenue Stillorgan Industrial Park Blackrock Co. Dublin Ireland Page 2

3 INDEX 1 INTRODUCTION AND PRINCIPLE KIT CONTENTS AND STORAGE ABOUT THE ARROW/LIAISON IXT INSTRUMENT Intended Users Installation and Calibration Technical Support RUNNING THE ARROW/LIAISON IXT INSTRUMENT CLEANING THE ARROW/LIAISON IXT INSTRUMENT PREPARING SAMPLES Cultured cells using trypsination Cultured cells using cell scraper Buccal cells (using Whatman Sterile Omni Swab or other swab types) Saliva (using Genotek Oragene DNA sample collection kit) Animal tissue TROUBLESHOOTING GUIDE PRODUCT LIST Page 3

4 1 INTRODUCTION AND PRINCIPLE DNA Extraction is intended for automated purification of genomic DNA directly from cultured cells and tissue, buccal swab, and saliva. It can be used on both the Nordiag Arrow Instrument and the LIAISON IXT instrument. The automation of DNA extraction reduces hands-on time, minimizes human errors, and increases sample throughput (1-12 samples per run). The principle of the purification procedure is as follows: Lysis buffer is added to the sample to release nucleic acids (NA). The nucleic acids are then bound to magnetic beads. After washing off sample material, the purified NA is eluted off beads and transferred to a storage tube. Page 4

5 2 KIT CONTENTS AND STORAGE Each DNA Extraction kit contains: 96 Cartridges for the DNA isolation procedure 98 Pumps 96 Tips 24 ml DNA Pretreatment Buffer 1 48 ml DNA Pretreatment Buffer 2 1 ml Proteinase K solution 1 Instructions for Use (IFU) WARNING: The cartridge contains guanidine thiocyanate, isopropanol and ethanol. The guanidine thiocyanate can form hazardous compounds if combined with bleach. Materials required but not provided Piercing tool Sample tubes (see Table 1) Elution tubes (optional brand) Commercial liquid household bleach, 5.25 % hypochlorite solutions or equivalent for sterilization of the instrument. Table 1: Recommended sample tubes for use with Arrow/LIAISON IXT. The table only lists the tubes tested at DiaSorin. Brand Compatible tubes Incompatible tubes Axygen Microtubes (MCT-series) Screw cap microtubes (ST-series) Corning Microtubes Costar Snap Cap (e.g. 3620) Corning Eppendorf Microtubes Standard, Safe-Lock, LoBind None determined Qiagen Collection tubes (1.5 and 2.0 ml) None determined Sarstedt Microtubes (e.g ) and screw cap microtubes (e.g ) None determined Storage The kits are shipped at room temperature. All reagents and buffers can be stored at room temperature (15 25 C). Do NOT freeze the reagent cartridges, and do NOT expose to direct sunlight. Page 5

6 3 ABOUT THE ARROW/LIAISON IXT INSTRUMENT 1. Intended Users The Arrow/LIAISON IXT automated pipetting instrument (fig. 1), is an IVD approved NA isolation device with protocols for multiple nucleic acid extraction procedures. The instrument is intended for use by professionals that are trained in molecular biological techniques and in the use of Arrow/LIAISON IXT and respective kits. A piercing tool for opening of cartridges is required and provided with the instrument. Sample tubes and elution tubes are required but not provided (see Table 1: Recommended sample tubes for use with Arrow/LIAISON IXT) Figure 1: The Arrow instrument and LIAISON IXT instrument (the colour of the Arrow instrument may vary from that shown in the picture) 2. Installation and Calibration Please refer to the Operator s Manual and Installation Manual of the instrument for further information. The instrument can be calibrated by qualified personnel when appropriate. 3. Technical Support If an instrument problem occurs, first consult section 7 Troubleshooting Guide. Make notes of the error message(s), if any, and any unusual observations, prior to mailing support. Please use customercare@ie.diasorin.com as your first line of reporting support issues. The instrument should be connected to an uninterruptible power supply (UPS) to prevent sudden stops of the instrument during a run (in case of a power failure in the electrical system in the building where it is located). Page 6

7 4 RUNNING THE ARROW/LIAISON IXT INSTRUMENT The running procedures on the instrument are as follows: 1 Select protocol and volumes 2 Load pump with tip 3 Load cartridge 4 Pierce cartridge 5 Load sample 6 Load elution tube 7 Start the protocol 8 Remove the eluate from the instrument Loading instructions 2, 3, 5, and 6 appear on the instrument screen after the protocol and elution volume has been selected. Details of all steps are given below: 1. Select protocol and elution volume a. Turn the power button at the left back side on, and then push the ON button on the front left side (See Operator`s Manual for further instructions). b. Press `Continue` on the first screen to let the instrument initialize. c. Press `START PROTOCOL` on the Arrow/LIAISON IXT main menu. d. Select the DNA Extraction protocol. e. Choose elution volume. Page 7

8 2. Load pump with tip a. Assemble pump and tip Hold the pump and press it down into the tip while the tip is in the tip box (fig.2). Press down until the pump and tip are well connected and sealed. The physical distance between the pump and the rim of the tip must be in the range 0-3 mm (fig.3). Take a pump Push pump into tip Assembled pump-tip is placed in instrument Figure 2: How to assemble pump and tip. OK Not OK The physical distance between the pump and the rim of the tip must be 0-3 mm. Figure 3: Correct and incorrect pump-tip assembly. Page 8

9 b. Load the assembled pump-tip Place the pump with tip in the instrument according to fig Insert the pump upwards first. Open tip holder clip and insert the tip. The rim of the tip must be onto the top of the metal edge, see fig Close tip holder clip. Figure 4: Pump-tip positioning and tip clip. The rim of the tip is in direct contact with the metal edge. Figure 5: Correct positioning of tip. The rim of the tip is too high above the metal edge. Figure 6: Incorrect positioning of tip. Page 9

10 3. Load cartridge Place the cartridge into the rack. Press the cartridge firmly in place such that the entire longitudinal edge (fig.7) is in direct contact with the rack. The cartridge hole in the rack is intentionally close-fit to ensure that the cartridge stays in place during the run. Make sure that the front is secured by the lock on the cartridge (fig.7). Position the cartridges according to table 2. Front Cartridge lock Longitudinal edge Figure 7: Cartridge with front lock and longitudinal edge. Table 2: Positioning of cartridges in the Arrow/LIAISON IXT. Example is shown for the first 6 samples. Number of samples Positioning in the instrument (track number) , 6 3 5, 6, 7 4 5, 6, 7, 8 5 5, 6, 7, 8, 9 6 4, 5, 6, 7, 8, 9 Page 10

11 4. Pierce cartridge The piercing tool (fig.8) is used to pierce holes in the cartridge foil while the rack with the cartridge is positioned in the instrument (fig.12). a. Piercing procedure Pierce from the back to the front of the cartridge, with the piercing tool oriented according to figure 9. A complete piercing is best obtained by resolute and continuous movements throughout the procedure. Resolutely pierce the back well (figure 9), and, without pausing, roll the tool through the remaining wells (figure 10). This 2-step procedure should take approximately 1 second per cartridge. If piercing is not complete, roll the tool back to the starting point (figure 11). b. Piercing tool cleaning procedure Immediately after piercing has been completed, clean the piercing tool by rinsing the tool under tap water. Leave to dry until next use. In cases where further cleaning is desirable (e.g. with ethanol, or RNaseAway ), ensure that the piercing tool has become dry before use*. Treatment with chemicals such as RNaseAway necessitates a subsequent thorough rinse in nuclease-free water. The piercing tool may be autoclaved. *WARNING: If ethanol is used for cleaning, and piercing is performed before the tool has become completely dry, then remaining ethanol will dissolve the print ink on the cartridge foil, with a subsequent risk of contaminating the reagents with ink. Figure 8: Piercing tool and its orientation with regard to the cartridge. Page 11

12 A complete piercing is best obtained by firm and continuous movements throughout the procedure. Figure 9: Firmly pierce the back well. The 2-step procedure should take approximately 1 second per cartridge. Figure 10: Without pausing, roll the tool through the remaining wells. Figure 11: If piercing is not complete, roll the tool back to the starting point. Page 12

13 5. Load sample Put the sample tube into the adapter position (fig.12). See section 2 (Table 1) for tube brand, and section 6 for sample preparation. Please note that adapters for the sample tubes (fig. 13) are included with the instrument. Both types of adapter can be used for microtubes. 6. Load elution tube Put the elution tube (optional brand) into the elution tube position (fig.12). Cartridges Front Cartridge lock Sample tubes Elution tube snap cap holder (optional to use) Elution tubes Position 1, 2, 3, 4, etc. Figure 12: A loaded rack. Figure 13: Adapters for sample tube 7. Start the protocol Close the door and press `START`. 8. Remove the eluate from the instrument When `Protocol finished` is displayed on the screen, the eluate is ready for downstream analysis. Page 13

14 5 CLEANING THE ARROW/LIAISON IXT INSTRUMENT After the protocol has been run, remove and discard the sample preparation waste, including the pump-tips and cartridges. WARNING: The cartridge contains guanidine thiocyanate, isopropanol and ethanol. The guanidine thiocyanate can form hazardous compounds if combined with bleach. If necessary, clean the instrument as follows: 1. Run the UV decontamination protocol. 2. Remove the rack (fig. 14) from the instrument. 3. Remove the magnet (fig. 15) 4. If required, the heating block (fig. 16) can be lifted for cleaning. NOTE: The heating block is connected to the instrument with a cable and cannot be removed entirely. 5. Clean the rack, the magnet and the inside of the instrument with detergent or alcohol depending on the material spilt. 6. See the Operator s Manual for further cleaning instructions. Figure 14. The Arrow/ LIAISON IXT rack Figure 15. The magnet Figure 16. The heating block Page 14

15 6 PREPARING SAMPLES 1. Cultured cells using trypsination a. Pour off medium from the culture flask, and wash the cell layer with PBS-EDTA. b. Add PBS-EDTA and leave at room temperature in the cell culture hood for 2-5 min. c. Pour off PBS-EDTA, and wash with trypsin. d. Add trypsin (enough to cover the cell layer) and leave in the growth incubator until the cells have detached from the plastic. Check in microscope. e. Resuspend the cells in PBS (or growth medium, if the cells aggregate when using PBS) and transfer the cell suspension to a 50 ml tube. f. Count the cells. g. Centrifuge at 1500 rpm for 10 min, and carefully resuspend the cell pellet in PBS to a concentration of ~2x10 6 /ml. Keep the cells on ice during the next steps. h. Count the cells again, and aliquot 1x10 6 cells per microtube. Centrifuge at full speed for 25 s. i. Remove the supernatant by aspiration (be careful not to disturb the cell pellet). j. For long-term storage of the cell pellets, freeze immediately in liquid nitrogen. Store at -80 C. k. To extract DNA from fresh or frozen cells, add a mix of 10 µl Proteinase K µl DNA Pretreatment Buffer 1 to each cell pellet as soon as possible. Vortex to loosen the cell pellet from the tube, and incubate at room temperature for 10 min. l. Put the sample tube (with lid off) in the Arrow/LIAISON IXT (fig.10). Note: If using microtube with snap cap, make sure that the cap stays clear of the tip when it comes to aspirate the sample. 2. Cultured cells using cell scraper a. Aspirate as much medium as possible from the plate, dish, or flask. b. Add DNA Pretreatment Buffer 1 to the cell layer, e.g. 170 µl to a 100 mm culture dish, or 240 µl to a 6- hole culture plate. c. Scrape the cells off using e.g. a 25 mm cell scraper, and transfer the ~250 µl sample to a microtube. d. Add 10 µl of Proteinase K to the sample. Cap the tube and vortex, before incubating the sample at room temperature for 10 min. e. Put the sample tube (with lid off) in the Arrow/LIAISON IXT (fig.10). Note: If using microtube with snap cap, make sure that the cap stays clear of the tip when it comes to aspirate the sample. Page 15

16 3. Buccal cells (using Whatman Sterile Omni Swab or other swab types) The following procedure is for buccal cell samples collected with Omni Swab. This involves the rubbing of the swab against the inside of the cheek 5-6 times, followed by ejection of the collection pad into an empty microtube and closing the lid. Cotton-tipped swabs may also be used, with obvious and minor adjustments of the procedure below. a. Add 500 µl of DNA Pretreatment Buffer 2 to the Omni Swab collection pad in the tube. Invert a few times to wet the swab completely. Due to the absorbent material in the Omni Swab, the 500 µl starting volume will be reduced to ~250 µl. b. Add 10 µl of Proteinase K to the sample tube containing the swab. Cap the tube and vortex at intermediate speed, before incubating the sample at 56 C for 10 minutes. Remove the swab using a pipette tip. c. Put the sample tube (with lid off) in the Arrow/LIAISON IXT (fig.10). Note: If using microtube with snap cap, make sure that the cap stays clear of the tip when it comes to aspirate the sample. 4. Saliva (using Genotek Oragene DNA sample collection kit) a. Pipette 250 µl Oragene DNA sample (see manufacturer`s instructions) into a sample tube and incubate at 56 C for 2 hrs or longer. b. Put the sample tube (with lid off) in the Arrow/LIAISON IXT (fig. 10). Note: If using microtube with snap cap, make sure that the cap stays clear of the tip when it comes to aspirate the sample. 5. Animal tissue a. Prepare a solution containing 10 µl Proteinase K µl DNA Pretreatment Buffer 2 for every 10 mg of tissue (and accordingly, e.g. 20 µl Proteinase K µl DNA Pretreatment Buffer 2 per 20 mg of tissue). b. As soon as possible, add 210 µl of the proteinase K + buffer solution per 10 mg of tissue (fresh or frozen). Cap tubes and vortex, before incubating the samples at 56 C for 2 hours to overnight, depending on tissue type. c. Transfer the sample ( µl) to a new tube, avoiding residual solid tissue (spin if necessary). d. Put the sample tube (with lid off) in the Arrow/LIAISON IXT (fig. 10). Note: If using microtube with snap cap, make sure that the cap stays clear of the Arrow/LIAISON IXT tip when it comes to aspirate the sample. Page 16

17 7 TROUBLESHOOTING GUIDE Problem Comments Low or zero eluate volume The amount of nucleic acid in the sample could be too high. Reduce the amount of sample material, while keeping sample volume at 250 µl and using the highest elution volume possible. The cartridge might not have been firmly in place in the rack during the run. For detailed instructions, see section 4.3 Load cartridge. The calibration settings could be out of range. Calibrate the instrument (see the Operator`s and Installation Manuals). If the eluate volume is low or zero despite satisfactory calibration settings, check the used cartridge against the photo below. If there is poor match, photograph the used cartridge from the top and the side and it to for further troubleshooting. DNA cartridge post-run Low yield from tissue The tissue piece could be too massive (tissue type dependent) for the proteinase K to digest properly. Cut the tissue piece into smaller units. The ratio of tissue weight to buffer volume could be too high. Weigh the tissue, and make sure that the sample contains 200 µl buffer and 10 µl proteinase K for each 10 mg of tissue. Visible amounts of beads in the eluate The presence of beads will affect absorbance readings, but not PCR or most other downstream analyses. To avoid bead influenced absorbance readings, remove beads by placing the eluates onto a magnet. Non-responsive instrument screen Disconnect the power cord, then reconnect to let the instrument re-initialize. If this does not help, contact DiaSorin at customercare@ie.diasorin.com. Instrument error message See the Arrow/LIAISON IXT Operator`s Manual. Page 17

18 8 PRODUCT LIST Table 3: Product list for ordering Arrow/LIAISON IXT kits. Prod. Code Product Description Size Arrow Instrument, CE/IVD A compact, automated user-friendly solution for nucleic acid extractions, running 1-12 samples. 1 piece I0074 LIAISON IXT Instrument, CE/IVD A compact, automated user-friendly solution for nucleic acid extractions, running 1-12 samples. 1 piece DNA Extraction Kit RNA Extraction Kit CellSep Kit CellSep Advanced Kit BUGS n BEADS Kit, CE/IVD Stool DNA Extraction Kit, CE/IVD Blood DNA 200 Extraction Kit, CE/IVD Blood DNA 500 Extraction Kit, CE/IVD Viral NA Extraction Kit, CE/IVD Other Supporting Products For isolation of genomic DNA from cultured cells, tissue, buccal swab and saliva. Includes cartridges, 2 buffers, proteinase K, tips and pumps For isolation of intact RNA from cultured cells. Includes cartridges, 1 buffer, tips and pumps. For isolation of cells from whole blood or buffy coat. Includes cartridges, sample tubes, tips and pumps. For isolation of 1-3 cell types from whole blood or buffy coat. Includes cartridges, sample tubes, tips and pumps. For isolation of mainly bacterial NA from various sample types, such as cell culture, urine, swab, and sputum. Includes cartridges, tips and pumps. For isolation of genomic, bacterial and viral DNA from human and animal stool. Includes cartridges, buffer, tips and pumps. For isolation of mainly genomic DNA from whole blood. Includes cartridges, tips and pumps. For isolation of mainly genomic DNA from whole blood, and buffy coat. Includes cartridges, tips and pumps. For isolation of viral NA from serum, plasma, swabs, and blood. Includes cartridges, tips and pumps. 96 preps 96 preps 96 preps 96 preps 96 preps 96 preps 96 preps 96 preps 96 preps tips in box Extra box of 96 tips 1 x 96 tips pumps in plastic bag Extra bag of 96 pumps 1 x 96 pumps Piercing tool For piercing of cartridges 1 piece Page 18

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20 DiaSorin Ireland Ltd. Unit 13/14 Holly Avenue Stillorgan Industrial Park Blackrock Co. Dublin Ireland Tel: +353 (1) Fax: +353 (1) Page 20