Version No.: 2.0 ( )
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2 Kit for the extraction of total RNA from wide range of tissue using MagListo Version No.: 2.0 ( ) Please read all the information in booklet before using the unit Bioneer Corporation 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: Fax: MagListo is a trademark of Bioneer Corporation. Copyright Bioneer Corporation. All Rights Reserved.
3 Contents I. Overview 1 II. Kit Components 1 III. Storage 2 IV. Intended Use 2 V. Safety Warnings and Precautions 2 VI. Warranty and Liability 2 VII. Technical Assistance 3 VIII. Quality Management 3 IX. Kit Specifications 4 Extraction of tissue total RNA from small amount of sample 4 Recommended amounts of starting sample 4 X. Sample Preparation 5 XI. Principle 5 XII. Magnetic Nano Bead Information 6 XIII. Guidelines for MagListo Magnetic Separation Rack 6 XIV. Materials and Equipment Needed But Not Provided 7 Types of the Magnetic Separation Rack 7 XV. Procedure 8 XVI. Protocols 9 Before you begin 9 A. RNA Extraction from Animal Tissue 9 B. RNA Extraction from Cultured Cells 14 C. ONE-Step RNA Cleanup (DNase Treatment) 18
4 D. RNA Cleanup (RNA Purification) 20 XVII. Appendix 21 Troubleshooting Guide 21 Experimental Data 23 XVIII. Ordering Information 25 XIX. Explanation of Symbols 26
5 I. Overview Description MagListo 5M Tissue Total RNA Extraction Kit utilizes Magnetic Nano Beads to extract total RNA from various of sources, such as animal tissue or cultured cells, with the aid of the MagListo Magnetic Separation Rack. The use of MagListo Magnetic Separation Rack along with the kit greatly increases user convenience by shortening the extraction time without centrifugation. Features and Benefits - Magnetic Nano Beads enable the rapid nucleic acid extraction - No requirement of expensive instruments - A single kit serves mini or midi scale experiment Applications Applicable to RNA extraction step for assays requiring RNA, including, but not limited, RT-PCR, cdna synthesis, Northern, dot, and slot blot analyses, Microarrays, and RNAseq II. Kit Components MagListo 5M Tissue Total RNA Extraction Kit * K-3613 Buffer 1 (Binding) 25 ml x 2 ea Buffer 2 (1 st Washing) 100 ml x 1 ea Buffer 3 (2 nd Washing) 100 ml x 1 ea Buffer 4 (3 rd Washing) 120 ml x 1 ea Buffer 5 (Elution) 20 ml x 1 ea Magnetic Nano Beads - RNA 1.8 ml x 6 ea *Mini 100 rxn, Midi 10 rxn 1
6 III. Storage MagListo 5M Tissue Total RNA Extraction Kit should be stored dry at room temperature. It can be stored for up to 2 years if it remains sealed. IV. Intended Use MagListo 5M Tissue Total RNA Extraction Kit is intended for research use only. This kit is not intended for human or veterinary diagnostics. V. Safety Warnings and Precautions Please inquire BIONEER s Customer Service Center to obtain a copy of the Material Safety Data Sheet (MSDS) for this product. Before, during and after using this kit, all potentially hazardous materials (i.e. materials that may have come in contacted with genetically recombinant samples) including tubes, tips and other kit contents should be processed and discarded in accordance with applicable and appropriate regulations of the municipality/government in which this product is being used. A user must also be equipped with basic experimental techniques required for correct execution of the extraction experiments described in this User s Guide. Some inventions applied in this kit may infringe existing patents in certain countries. The purchase of this kit does not include or provide a license to perform such patented inventions. Users may be required to obtain a license depending on the patent law of the country where this product is being used. We do not condone nor recommend the unlicensed use of patented inventions. VI. Warranty and Liability All BIONEER products are manufactured and tested under strict quality control protocols. BIONEER guarantees the quality of all directly manufactured products during the warranty period of one (1) year from the date of purchase. If you find any issues regarding the product quality, please immediately contact BIONEER s Customer Service Center (sales@bioneer.com). BIONEER does not assume any liability for the misuse of the product, i.e. using the product for any 2
7 purposes other than its intended purpose as described in the User s Guide. BIONEER will only assume liability under the condition when the users disclose all related information regarding the issue to BIONEER in written form within 30 days after occurrence of the issue. VII. Technical Assistance At Bioneer, we pride ourselves on being responsive to your needs. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in Molecular Biology and the use of Bioneer products. If you have any questions or would like to find out more information about MagListo products, please contact us. We look forward to hearing from you! Technical Support For all technical questions and troubleshooting on Bioneer products and applications Tel: sales@bioneer.com - In North America Tel: support@bioneer.us.com VIII. Quality Management Every aspect of our quality management system from product development to supplier qualification ensures that our products meet the world-class standards. Each lot of MagListo 5M Tissue Total RNA Extraction Kit is carefully tested by the quality control team. 3
8 IX. Kit Specifications Mini scale Midi scale Starting sample 20 mg tissue or 3 x 10 6 cells 200 mg tissue or 2 x 10 7 cells Extraction time < 10 min < 15 min Minimum elution volume 50 µl 500 µl Expected purity A 260/280 > 1.9 *RNA content can vary greatly according to tissue type. Extraction of tissue total RNA from small amount of sample MagListo 5M Tissue Total RNA Extraction Kit is also able to extract total RNA from a small quantity of sample. Refer to RNA Extraction from Cultured Cell for Mini in page 14 for more details about RNA extraction from samples with a low number of cells ( 3x10 6 ). Recommended amount of starting sample It is recommended to use the amounts in Table 1 as starting sample amount. Table1. Growth area and Average cell yield in various culture dishes. Cell culture dishes Growth area (cm 2 ) Average cell yield Multi well plate 6 well x well 4 4 x well 2 2 x well 1 1 x well x 10 4 Dishes 35 mm x mm 21 3 x mm 55 8 x mm x 10 7 Flasks 50 ml x ml 75 1 x
9 X. Sample Preparation Several factors, such as harvesting method and storage of starting samples can influence the yield and purity of RNA. All specimens must be stored in a -70 freezer or used immediately after collection. It is recommended to put the sample as soon as possible on ice, and to avoid repeated freezing and thawing. Repeating freeze-thawing of the sample will result in the degradation of RNA. Tissue Tissue samples should immediately be used or stored at -70 for optimal results. To disrupt tissue sample, grind it in a pestle and a mortar with liquid nitrogen. Alternatively, a homogenizer or a bead-beater can be used. Cultured cells Cultured cells can easily be harvested using a centrifuge. However, it might be difficult to extract total RNA if cultured cells are too clustered. In this case, trypsin can be used to scatter cells from the cluster. For optimal extraction, the number of cells should be less than 2 x 10 7, which is calculated with a cell counter. It is recommended to keep samples on ice before use. XI. Principle The MagListo 5M Tissue Total RNA Extraction Kit is designed for the extraction of high purified total RNA from tissue and cultured cells. The overall principle is based on adsorption of RNA onto the Magnetic Nano Bead by chaotropic salt. For example, chaotropic agents in Buffer 1 (Binding) contains guanidine hydrochloride and guanidine thiocyanate, as which remove water molecules around RNA and silica coated magnetic beads surface resulting in RNA then being captured by magnetic beads. The Magnetic Nano Beads and RNA complexes are pulled and fixed on the tube wall using a magnetic force, followed by washing with ethanol to remove debris and excessive salts. Finally, the captured RNAs are then eluted by Buffer 5 (Elution), an aqueous solution with optimal ph. Sample Lysis Binding Washing Elution 5
10 XII. Magnetic Nano Bead Information Description Magnetic Nano Beads have been developed to overcome shortcomings of existing resin and to automate purification process. The principle of extraction using the Magnetic Nano Beads is that coated functional group on the surface of the Magnetic Nano Beads bind with DNA and the Magnetic Nano Beads are then isolated using external magnetic field. Features Fast binding guarantees higher throughput automation Large surface area enables more sensitive assay Globular structure increases specificity by decreasing non-specific binding Specification Matrix Silica-coated Fe 3 O 4 AccuNanoBead TM Silica Magnetic Nano Beads Average size Ligand 400nm -OH Working Temp. 0~100 Storage Store at room temperature upon receipt XIII. Guidelines for MagListo TM Magnetic Separation Rack Description MagListo Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic Nano Beads. These racks of different sizes allow users to choose the product according to their needs. The following are recommended when handling the MagListo Magnetic Separation Rack The product is made of acryl and plastic. Be careful not to drop the product as the dropping may break the product. When moving the product, take extra care not to drop the product as it may cause injury. If the product is broken, do not discard it with bare hands as the sharp edges may cause injury. 6
11 When an extracted or purified nucleic acid is spilled on the product, immediately rinse it with running water and clean it with 70% ethanol. Acetone, Toluene, or organic solvent may damage the acrylic and plastic part of the product, which may lead to malfunction of the product. Rinse the product immediately when spillage of any above mentioned solvents occurs as the expected DNA yield may not be obtained if the product is damaged. Make sure that a corrosive liquid on the magnet plate part of the product. If spillage occurs, immediately rinse it off with running water as it may corrode the magnet during storage and may degrade its performance. XIV. Materials and Equipment Needed But Not Provided 1. Table-top microcentrifuge, 16,000 x g (> 13,000 rpm) (mini scale) 2. Centrifuge with rotor capable of 3,000 x g (midi) ml or 2 ml tube (mini scale) ml tube, 50 ml tube (midi scale) 5. Vortex mixer 6. Absolute ethanol 7. Thermal block or dry oven 8. MagListo Magnetic Separation Rack Types of the Magnetic Separation Rack Tube MagListo Magnetic Separation Rack Cat.no 1.5 ml or 2 ml microcentrifuge tube MagListo -2 Magnetic Separation Rack TM ml tube MagListo -15 Magnetic Separation Rack TM ml centrifuge tube MagListo -50 Magnetic Separation Rack TM-1030 (Note) Please refer to the ordering information in this User s Guide for more information regarding catalog number of racks designed for specific size of tubes. 7
12 XIV. Procedure-Tissue Total RNA Extraction 8
13 XVI. Protocols Before you begin 1. Buffer 1 (Binding) contains chaotropic salt. You should take appropriate laboratory safety precautions and wear gloves and lab goggles when handling. 2. The relative centrifugal force (RCF) is calculated in g as follows: RCF = 1.12 x r x (rpm/1,000) 2 Where r is the radius of a rotor in cm, and rpm is the speed of the rotor in revolutions per minute. 3. To inhibit RNase activity, we recommend adding β-mercaptoethanol to Buffer 1 (Binding) before use. Add 10 µl β-mercaptoehanol (>99%) per 1 ml Buffer 1 (Binding). A. RNA Extraction from Animal Tissue for Mini/Midi Scale 1. (Tissue lysis & homogenization) Disruption and homogenization using a rotor-stator homogenizer : Place the weighed (fresh, frozen, or RNAlater -stabilized) tissue (~20 mg (mini) / ~200 mg (midi)) in a suitable-sized vessel. Add 400 µl (mini) / 4 ml (midi) of Buffer 1 (Binding) to the tissue sample. Immediately disrupt and homogenize the tissue using a conventional rotor-stator homogenizer until it becomes uniformly homogeneous and transfer it to a new 2 or 1.5 ml tube (go to step 4). 2. Disruption using a mortar and pestle followed by homogenization using a needle and syringe: Immediately place the weighed (fresh, frozen, or RNAlater -stabilized) tissue in liquid nitrogen, and grind the sample thoroughly with a mortar and pestle. Decant tissue powder and liquid nitrogen into an RNase-free, liquid-nitrogen-cooled 2 ml tube. Allow the liquid nitrogen to evaporate, but do not allow the tissue to be thawed (go to step 3). 3. Add 400 µl (mini) / 4 ml (midi) of Buffer 1 (Binding) to each tube and mix thoroughly using a vortex mixer. Make sure that the sample is completely resuspended the sample to achieve maximum lysis efficiency. (Note) Insufficient homogenization can decrease the yield of total RNA purified, and also may cause clogging of Magnetic Nano Beads in the following steps. For a sufficient homogenization of the lysate, make the lysate, pass through a blunt 20-gauge needle (0.9 9
14 mm diameter) 5 to 10 times. A. (Mini) please transfer the lysate to a 1.5 ml or 2 ml tube. B. (Midi) please transfer the lysate to a 50 ml tube. 4. (RNA precipitation) Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to each tube and mix well using a vortex mixer or by pipetting. 5. (RNA binding with Magnetic Nano Bead: 5-7) Add 100 µl (mini) / 1 ml (midi) of Magnetic Nano Beads solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully resuspend. (Note) Please shake the Magnetic Nano Bead solution well or mix completely with a vortex mixer before use. 6. Place the tube in MagListo -2 (mini) / MagListo -50 (midi) Magnetic Separation Rack with the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to magnet. - Attachment of the magnet plate Combine the magnet plate to the stand. 7. Without removing the tube from MagListo Magnetic Separation Rack, discard the supernatant carefully and completely remove the remaining supernatant on a paper towel by blotting action. During this process, the magnet crude pellet remains attached to the side of tube. (Optional) If performing optional ONE Step RNA Clean up, follow steps (page 12) after performing this step. - How to discard the supernatant Discard the supernatant by inverting the MagListo Magnetic Separation Rack. The silicone 10
15 immobilizer inside the stand prevents the tubes from falling. When discarding the supernatant, invert the rack completely so that the solution does not spill on the rack. 8. (1 st Washing: 8-10) Detach the magnet plate from MagListo Magnetic Separation Rack. Add 1ml (mini) / 10 ml (midi) of Buffer 2 (1 st Washing) to the each tube and close the cap. Mix with a vortex mixer until the beads are fully resuspended. - Detachment of the magnet plate Detach the magnet plate gently by pulling it upwards. 9. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 10. Without removing the tube MagListo Magnetic Separation Rack, discard the supernatant and remove the remaining supernatant on a paper towel by blotting action. 11. Repeat the steps 8 through 10 by adding 1 ml (mini) / 10 ml (midi) of Buffer 3 (2 nd Washing) instead of Buffer 2 for additional washing. 12. (3 rd Washing) Without removing the tube from MagListo Magnetic Separation Rack, add 1 ml (mini) / 10 ml (midi) of Buffer 4 (3 rd Washing) to the opposite side of bead pellet. Close the cap and invert the rack twice in order to remove ethanol from the sample. (Note) Direct pipetting of Buffer 4 onto the bead pellet, vortexing and/or vigorous shaking of the tubes may release nucleic acid from the beads, which may result in lower RNA yield than expected. 11
16 13. Discard the supernatant and completely remove the remaining supernatant by blotting action. - Add Buffer 4 and discard the supernatant 14. (Elution: 14-18) Detach the magnet plate from the MagListo TM Magnetic Separation Rack. Add μl (mini) / 500 μl-1 ml (midi) of Buffer 5 (Elution) to the tube with the magnet plate detached and resuspend RNA by vortexing or pipetting. 15. Incubate the tube at for 1 min. 16. Attach the magnet plate to MagListo Magnetic Separation Rack and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 17. Without removing the tube from MagListo Magnetic Separation Rack, transfer supernatant containing RNA carefully to a new sterile microcentrifuge tube. 18. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads. 12
17 Summary of reagent volumes required in each step of Tissue Total RNA Extraction Step Buffer Mini scale Midi scale Tissue Lysis Buffer 1 (Binding) 400 μl 4 ml RNA precipitation Absolute ethanol 200 μl 2 ml RNA Binding Magnetic Nano Beads - RNA 100 μl 1 ml 1 st Washing Buffer 2 (1 st Washing) 1 ml 10 ml 2 nd Washing Buffer 3 (2 nd Washing) 1 ml 10 ml 3 rd Washing Buffer 4 (3 rd Washing) 1 ml 10 ml Elution Buffer 5 (Elution) μl 500 μl - 1 ml 13
18 B. RNA Extraction from Cultured Cells in Mini/Midi Scale 1. (Harvest cell) Cells grown in suspension: Count the cell number, then centrifuge given number of cells (~3x10 6 (mini) / 2x10 7 (midi)) at 300 x g for 5 min. Discard supernatant carefully and go to lysis & homogenization (go to step 3). 2. Cells grown in a monolayer: There are 2 different ways to collect cells grown in a monolayer. a. Direct cell lysis on the Culture Dish: Completely remove Cell Culture Medium and go to lysis & homogenization (go to step 3). (Remaining medium may inhibit with the RNA extraction) b. Harvesting cells with trypsin: Remove Cell Culture Medium and wash the monolayer with DPBS. Add 0.1%-0.25% typsin to the washed cell monolayer. When the cells are detached, add Cell Culture Medium to inactivate the typsin. Transfer the cells into a RNase-free tube and centrifuge at 300 x g for 5 min. Discard supernatant carefully and go to lysis & homogenization (go to step 3). 3. (Lysis & homogenization) Add 400 µl (mini) / 4 ml (midi) of Buffer 1 (Binding) to each tube and mix thoroughly using a vortex mixer. Make sure that you must completely resuspend the sample to achieve maximum lysis efficiency. (Note) Insufficient homogenization can decrease the RNA purification yield, and also cause clogging of Magnetic Nano Beads in the following steps. For the sufficient homogenization of the lysate, make the lysate passed through blunt 20-gauge needle (0.9 mm diameter) 5 to 10 times. A. (Mini) please transfer the lysate to a 1.5 ml or 2 ml tube. B. (Midi) please transfer the lysate to a 50 ml tube. 4. (RNA precipitation) Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to the each tube and mix well using a vortex mixer or by pipetting. 5. (RNA binding with Magnetic Nano Bead: 5-7) Add 100 µl (mini) / 1 ml (midi) of Magnetic Nano Beads solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. 14
19 (Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix with a vortex mixer before use. 6. Place the tubes on the MagListo -2 (mini) / MagListo -15 (midi) Magnetic Separation Rack with the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. - Attachment of the magnet plate Combine the magnet plate to the stand. 7. Without removing the tube form MagListo Magnetic Separation Rack, discard the supernatant carefully and completely remove the remaining supernatant on a paper towel by blotting action. (Optional) If performing optional ONE Step RNA Cleanup (DNase Treatment), follow steps (page 12) after performing this step. - How to discard the supernatant - Discard the supernatant by inverting the MagListo Magnetic Separation Rack. The silicone immobilizer inside the stand prevents the tubes from falling. When discarding the supernatant, invert the rack completely so that the solution does not to spill on the rack. 8. (1 st Washing: 8-10) Detach the magnet plate from MagListo Magnetic Separation Rack. Add 1 ml (mini) / 10 ml (midi) of Buffer 2 (1 st Washing) to the each tube and close the cap. Mix by vortexing or shaking until the beads are fully resuspended. 15
20 - Detachment of the magnet plate Detach the magnet plate gently by pulling it upwards. 9. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 10. Without removing the tubes from the MagListo Magnetic Separation Rack, discard the supernatant and remove the remaining supernatant on a paper towel by blotting action. 11. (2 nd Washing) Repeat the steps 8 through 10 by adding 1 ml (mini) / 10 ml (midi) of Buffer 3 (2 nd Washing) instead Buffer 2 for additional washing. 12. (3 rd Washing) Without removing the tubes from the MagListo Magnetic Separation Rack, add 1 ml (mini) / 10 ml (midi) of Buffer 4 (3 rd Washing) to the opposite side of bead pellet. Close the cap and gently invert the rack twice in order to remove ethanol from the sample. (Note) Direct pipetting of Buffer 4 onto the bead pellet, vortexing and/or vigorous shaking of the tubes may release nucleic acid from the beads, which may result in lower RNA yield than expected. 13. Discard the supernatant and completely remove the remaining supernatant by blotting action. - Add Buffer 4 and discard the supernatant 16
21 14. (Elution: 14-18) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add μl (mini) / 500 μl-1 ml (midi) of Buffer 5 (Elution) to the tube with the magnet plate detached and resuspend by vortexing or pipetting or vortex mixer for 15 sec. 15. Incubate the tube at for 1 min. 16. Place the tubes on the MagListo Magnetic Separation Rack. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 17. Without removing the tubes from the MagListo Magnetic Separation Rack, transfer supernatant containing DNA supernatant to a new sterile microcentrifuge tube. 18. Discard the tubes with remaining Magnetic Nano Bead pellet. Do not reuse the beads. Summary of reagent volumes required in each step of Cell Total RNA Extraction Step Buffer Mini scale Midi scale Cell Lysis Buffer 1 (Binding) 400 μl 4 ml RNA precipitation Absolute ethanol 200 μl 2 ml RNA Binding Magnetic Nano Beads - RNA 100 μl 1 ml 1 st Washing Buffer 2 (1 st Washing) 1 ml 10 ml 2 nd Washing Buffer 3 (2 nd Washing) 1 ml 10 ml 3 rd Washing Buffer 4 (3 rd Washing) 1 ml 10 ml Elution Buffer 5 (Elution) μl 500 μl - 1 ml 17
22 C. ONE Step RNA Clean Up (DNase Treatment) 1. (RNA precipitation) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 1 ml (mini) / 10 ml (midi) of absolute ethanol to the each tube and close the cap. Mix by vortexing or shaking until the beads are fully resuspended. 2. Place the tubes on the MagListo -2 (mini) / MagListo -50 (midi) Magnetic Separation Rack with the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 3. Without removing the tubes from the MagListo Magnetic Separation Rack, discard the supernatant carefully and completely remove the remaining supernatant using a paper towel by blotting action. 4. The beads can be dried with a dry oven at 65 for following times. (mini: > 5 min, midi: >15 min) Please use a clean bench during the drying procedure to prevent RNase or other aerosol contamination. 5. Add DNase Reaction Buffer and RNase-Free DNase to the each tube. (mini: up to 70 µl, midi: up to 700 µl) 6. Detach the magnet plate from the MagListo Magnetic Separation Rack and close the cap. Mix with a vortex mixer until the beads are fully resuspended. 7. Place on the benchtop (20 30 C) for 20 min. 8. Detach the magnet plate from MagListo Magnetic Separation Rack. Add 300 µl (mini) / 3 ml (midi) scale extraction of Buffer 2 (1 st Washing) and 300 µl (mini) / 3 ml (midi) scale extraction of absolute ethanol to the each tube. Close the cap and mix with a vortex mixer until the beads are fully resuspended. 9. Place the tubes on the MagListo Magnetic Separation Rack with the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 18
23 10. Without removing the tubes from the MagListo Magnetic Separation Rack, discard the supernatant carefully and completely remove the remaining supernatant using a paper towel by blotting action. 11. Go to step 11 of A. RNA Extraction from Animal Tissue in page 11 and follow the instructions accordingly. 19
24 D. RNA Clean up (RNA Purification) 1. Transfer RNase-free water into RNA sample to a volume of 100 µl. (Optional) If you DNA-free RNA is required, add DNase Reaction Buffer and RNase-Free DNase to each tube and make the volume up to 100 µl with RNase-free water. Incubate the tubes for 10 min at room temperature. 2. Add 100 µl of Buffer 1 (Binding) to each tube and mix completely using a vortex mixer. 3. Add 200 µl of absolute ethanol to each tube and mix completely using a vortex mixer. 4. Add 100 µl of Magnetic Nano Beads solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. (Note) Magnetic Nano Bead Solution contains Magnetic Nano Beads. Please shake well or mix with a vortex mixer before use. 5. Place the tubes on the MagListo -2 (mini) Magnetic Separation Rack with the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to magnet. 6. Without removing the tubes from the MagListo Magnetic Separation Rack, discard the supernatant carefully and completely remove the remaining supernatant on a paper towel by blotting action. 7. Go to step 8 of A. RNA Extraction from Animal Tissue in page 11 and follow the instructions accordingly. 20
25 XV. Appendix Troubleshooting guide This troubleshooting guide will help you to solve problem that may arise during RNA extraction. For other technical assistance or more information, please contact our technical assistance team. Comments and suggestions Buffers or other reagents may have been exposed to external factors that may have reduced its quality. Please make sure that reagents are stored at room temperature at all times upon arrival and that all reagent bottles are closed tightly, in order to preserve ph and stability, and to avoid contamination. Excess amount of starting sample was used to extract DNA. Appropriate amount of starting sample (see Kit Specification in page 4) should be used for efficient extraction of RNA. Elution may have been incomplete. Please extend incubation time up to 3 Low yield of RNA minutes at elution step to improve the yield. In addition, make sure that Magnetic Nano Beads are suspended completely in the eluting solution during incubation. Some of Magnetic Nano Bead pellet may have been lost while discarding solution. Check that all of the Nano Beads have bound tightly to the magnet when you discard supernatant. Insufficient shaking or vortexing during lysis step may lead to low DNA yield than expected. Shake or mix with a vortex mixer sufficiently during incubation step. Cell culture medium may have been incomplete. The best approach is to remove the medium as much as possible. Any leftover in the medium can lead to the inhibition of RNA extraction. 21
26 Beads may have been washed insufficiently. You must properly wash the beads in the 3 rd washing step. Remaining ethanol can decrease the purity of DNA. Take enough time to properly wash the beads. Low A 260/280 ratio Incomplete suspension of beads during the washing step causes salts to remain in the purified RNA. Make sure that the beads are suspended thoroughly during the washing process. Excessively clustered Magnetic Nano Bead Excess amount of starting sample is used to extract RNA. Appropriate amount of starting material (see Kit Specification in page 4) should be used for efficient extraction of RNA. Presence of a white precipitate in buffers A white precipitate may form in Buffer l (Binding) due to prolonged storage at low temperatures. Incubate Buffer l (Binding) at 60 to dissolve any precipitate in the buffer. RNase contamination can be degraded RNA. Use a heat gun or a blow dryer in a clean bench to prevent the contamination of RNase in the air. Use RNase-free pipette tips and change the gloves frequently. Degraded RNA Cultured cell samples that have been stored at -80 or lysis the samples with Buffer 1 (Binding) and then store at -80. Frequent freezing and thawing may result in lower RNA yield than expected. Avoid repeated freezing and thawing. Flotation of extracted DNA when loaded on an agarose gel Floating of RNA on an agarose gel is caused by the remaining ethanol in the eluted RNA. Ensure that the 3 rd Washing (ethanol removing) step in the protocol is properly performed. Remaining ethanol may also interrupt the enzymatic reaction.. 22
27 Experimental data Figure 1. Comparison of liver s Total RNA purified with MagListo 5M Tissue Total RNA Extraction Kit and competitor s kit (Single Column Type) MagListo MagListo Competitor Q 1-4: Extracted liver total RNA purified with Bioneer MagListo TM 5M Tissue Total RNA Extraction Kit 5-8: Extracted liver total RNA purified with competitor Q kit Sample Total Yield (ug) A260/A280 A260/A Figure 2. Comparison of kidney s Total RNA purified with MagListo 5M Tissue Total RNA Extraction Kit and competitor s kit (Single Column Type) : Extracted kidney total RNA purified with Bioneer MagListo TM 5M Tissue Total RNA Extraction Kit MagListo Competitor 5-8: Extracted kidney total RNA purified with competitor Q kit Sample Total Yield (ug) A260/A280 A260/A
28 Table1.Result of capillary electrophoresis of Total RNA extraction from HeLa cell using LabChip Sample RNA Quality Score 5S Area 5S %Total 18S Area 18S %Total 28S Area 28S %Total rrna Area Ratio [28S/18S] rrna Height Ratio [28S/18S] MagListo Competitor
29 XIV. Ordering Information Cat no. Product Description Size K-3601 MagListo TM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit K-3600 MagListo TM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit K-3603 MagListo TM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3605 MagListo TM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3607 MagListo TM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit K-3609 MagListo TM 5M PCR Purification Kit, 100 reactions (mini) 1 kit K-3611 MagListo TM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit K-3613 MagListo TM 5M Tissue Total RNA Extraction Kit, 100 reactions (mini) 1 kit K-3615 MagListo TM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit K-3617 MagListo TM 5M Viral DNA / RNA Extraction Kit, 100 reactions (mini) 1 kit K-3619 MagListo TM 5M Circulating Cell Free DNA Extraction Kit, 50 reactions (mini) 1 kit TM-1000 MagListo TM -8Ch Magnetic Separation Rack 1 ml tube x 8 holes TM-1010 MagListo TM -2 Magnetic Separation Rack 2 ml tube x 8 holes TM-1020 MagListo TM -15 Magnetic Separation Rack 15 ml tube x 6 holes TM-1030 MagListo TM -50 Magnetic Separation Rack 50 ml tube x 3 holes HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk HT-20-NG 2 ml microcentrifuge tube 500 ea / pk 25
30 XIX. Explanation of Symbols Catalog Number Batch code Manufacturer Contains sufficient for (n) tests Caution, consult accompanying documents Caution, Potential Biohazard USE BY Temperature Limitation DO NOT REUSE Consult Instruction For Use 26
31
Please read all the information in booklet before using the unit
100 Please read all the information in booklet before using the unit Bioneer Corporation 8-11,Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: +82-42-930-8777 Fax: +82-42-930-8688 Email:
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