MagListo 5M Tissue Total RNA Extraction Kit

Similar documents
Please read all the information in booklet before using the unit

E.Z.N.A. mirna Kit. R preps R preps R preps

Cells and Tissue DNA Isolation Kit (Magnetic Bead System) 50 Preps Product # 59100

Cytoplasmic & Nuclear RNA Purification Kit Product # 21000, 37400

Kit Components Product # (50 samples) Wash Solution A Elution Buffer B

MagListo 5M Nucleic Acid Extraction Kit MagListo Magnetic Separation Rack

E.Z.N.A. PF Micro RNA Kit. R preps R preps

Product # 24700, 24750

For simultaneous purification of genomic DNA and total RNA from the same animal cells or tissues

E.Z.N.A. Tissue RNA Kit. R preps R preps

Plant/Fungi Total RNA Purification Kit Product # 25800, 31350, 25850

Purification of cytoplasmic RNA from animal cells using the RNeasy Mini Kit

E.Z.N.A. Total RNA Kit II. R preps R preps R preps

HighPrep Blood & Tissue DNA Kit

Manual innuprep Micro RNA Kit

Plasmid Midiprep Plus Purification Kit. Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only

Total RNA Purification Micro Kit Product # 35300

SurePrep Nuclear or Cytoplasmic RNA Purification Kit

GenUP Virus DNA/RNA Kit

Note: for laboratory research use only. RNA High-purity Total RNA Rapid Extraction Kit (Spin-column) Signalway Biotechnology

Specifications. Kit Specifications. Alcohol Precipitation: Up to 100 ml Column Purification: Up to 5 ml Column Binding Capacity 25 µg

Kit for total DNA isolation from yeast

Average Yields* Yeast DNA Yeast RNA Time to Complete 10 Purifications * Yield will vary depending on the type of sample processed

RNAprotect Bacteria Reagent Handbook

ISOLATE II Blood DNA Kit. Product Manual

Presto Mini gdna Bacteria Kit

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

EZ-DNA. Instructions for Use. Genomic DNA Isolation Reagent. Product Description. Kit Reagent. Reagent Required But Not Supplied.

RNA Clean-Up and Concentration Kit Product # 23600, 43200

Kit Specifications 45 g. 45 g of RNA 8 L

Presto Mini RNA Bacteria Kit

Blood DNA Extraction Kit

Product # Kit Specification. Kit Specifications Maximum Column Binding Capacity 50 µg Maximum Column Loading Volume 650 µl

GenElute Total RNA Purification Kit. Catalog number RNB100

Geneaid DNA Isolation Kit (Yeast)

HiYield TM Genomic DNA Extraction Kit

E.Z.N.A. Total RNA Kit I. R preps R preps R preps

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

sbeadex maxi plant kit

Size-Select Kit for NGS Library Preparation Product # 53600

E.Z.N.A. HP Total RNA Kit. R preps R preps R preps

MD60002 MD MD62002

E.Z.N.A. Bacterial RNA Kit. R preps R preps

Absolutely RNA Microprep Kit

E.Z.N.A. Stool DNA Kit. D preps D preps D preps

AccuPrep 96 Genomic DNA Extraction Kit Cat. No.: K

ReliaPrep gdna Tissue Miniprep System

Product # Specifications. Kit Specifications Maximum DNA Input. 200 g Maximum DNA Volume Input Final Endotoxin Levels

User manual Invisorb Spin Plant Mini Kit

E.Z.N.A. Stool DNA Kit. D preps D preps D preps

UltraClean Midi Plasmid Prep Kit

Product # Specifications. Kit Specifications Maximum Column Binding Capacity (gdna) Maximum Column Loading Volume.

PureLink RNA Micro Kit

TIANamp Yeast DNA Kit

ISOLATE II Plant DNA Kit. Product Manual

E.Z.N.A. Water DNA Kit. D preps D preps D preps

PowerMag Soil DNA Isolation Kit (Optimized for KingFisher )

foodproof Sample Preparation Kit III

Introduction. Principle

E.Z.N.A. MicroElute Genomic DNA Kit. D preps D preps D preps

PowerSoil DNA Isolation Kit

ReliaPrep FFPE gdna Miniprep System

Lysis Buffer A Wash Solution A Elution Buffer B Mini Filter Spin Columns 50 Collection Tubes 50 Elution tubes (1.7 ml) 100 Product Insert 1

Urine DNA Isolation Maxi Kit (Slurry Format) Product # 50100

PowerMax Soil DNA Isolation Kit

Mag-Bind Stool DNA 96 Kit. M x 96 preps M x 96 preps

E.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps

Reagents provided. R075 RBC Lysis Buffer (10X RBL) 5 ml 25 ml 80 ml 160 ml

2 BQ Revision : 1( )

Product # (10 preps) Lysis Additive A Binding Buffer I. Elution Buffer B

FastGene Plasmid Mini Kit For isolation of high copy and low copy plasmid DNA

Mag-Bind Universal Pathogen 96 Kit. M x 96 preps M x 96 preps

QIAamp Fast DNA Stool Mini Handbook

PowerMag Soil DNA Isolation Kit

Unzipping Genes. MB566 RDP Trio TM Reagent. Product Code MB ml (100 ml RDP Trio TM Reagent) MB566-5 X 100 ml (5X100 ml RDP Trio TM Reagent)

TIANamp Marine Animals DNA Kit

SurePrep Soil DNA Isolation Kit

foodproof Magnetic Preparation Kit III

PureLink Plant Total DNA Purification Kit

E.Z.N.A. Blood DNA Maxi Kit. D preps D preps

PreAnalytiX Supplementary Protocol

mirneasy Mini Handbook

Supplementary File 3: DNA and RNA isolation

E.Z.N.A. Blood DNA Mini Kit. D preps D preps D preps

MasterPure Plant RNA Purification Kit

Absolutely RNA Nanoprep Kit

Manual innuprep Virus DNA Kit

BioSprint DNA Plant Handbook

FosmidMAX DNA Purification Kit

ULTRAPrep PLASMID DNA KIT

QIAfilter Plasmid Midi Kit (Cat #: 12243)

Mag-Bind Total Pure NGS. M ml M ml M ml

GeneMATRIX Gram Plus & Yeast Genomic DNA Purification Kit

For Research Use Only Ver

ab Plant Chromatin Extraction Kit

Reagent Kit Selection Guide

QIAamp DNA FFPE Tissue Handbook

MB542 HiPurA TM Soil DNA Purification Kit

QIAGEN GeneRead QIAact Panel Cleanup Kit Handbook

TIANamp Soil DNA Kit. For isolation of genomic DNA from soil.

Transcription:

User s Guide MagListo 5M Tissue Total RNA Extraction Kit K-3612 K-3613

MagListo TM 5M Tissue Total RNA Extraction Kit MagListo 5M Tissue Total RNA Extraction Kit Kit for the extraction of total RNA from a wide range of Tissue types using MagListo TM User s Guide K-3612 K-3613 8 100 Version No.: 1.1 (2016-11) 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: sales@bioneer.co.kr MagListo is a trademark of Bioneer Corporation. Copyright 2016. Bioneer Corporation. All Rights Reserved.

MagListo TM 5M Tissue Total RNA Extraction Kit 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 3 VII. Technical Assistance 3 VIII. Quality Management 3 IX. Product Specifications 4 X. Principle 4 XI. Materials and Equipment Needed But Not Provided 5 Choice of the right Magnetic Separation Rack 5 XII. Protocols Before you begin 6 RNA Extraction from Animal Tissue 7 RNA Extraction from Cultured Cells 9 ONE-Step RNA Cleanup 12 RNA Cleanup Protocol 13 XIII. Troubleshooting Guide 14 XIV. Ordering Information 15 XV. Explanation Symbols 16 -

I. Overview Description MagListo 5M Tissue Total RNA Extraction Kit is an innovative product to extract total RNA from a wide range of tissue types. Additionally this kit can be used for a sample of cultured cells. This kit is designed to be applicable to mini and midi scale extraction of tissue total RNA by using a proper volume of each solution suggested in this User s Guide. MagListo 5M Tissue Total RNA Extraction Kit provides high-quality RNA in unprecedented short period of time. MagListo Magnetic Separation Rack, which is also available from Bioneer (TM-1010 to 1030), will greatly enhance user s convenience and save processing time by removing the necessity of centrifugation and minimizing pipetting steps. Features and Benefits -Magnetic NanoBeads enable the rapid extraction of RNA as short as 10 min. -No need of expensive instruments. -One kit serves either mini or midi scale extraction. 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-3612 ** K-3613 Buffer 1 (Binding) 4 ml x 1 ea 25 ml x 2 ea Buffer 2 (1 st Washing) 5.5 ml x 1 ea 55 ml x 1 ea Buffer 3 (2 nd Washing) 2.0 ml x 1 ea 20 ml x 1 ea Buffer 4 (Elution) 1.8 ml x 1 ea 25 ml x 1 ea Magnetic NanoBeads - RNA 1 ml x 1 ea 1.8 ml x 6 ea *mini - 8 RXNs, midi 1 RXN **mini 100 RXNs, midi 10 RXNs 1

III. Storage MagListo 5M Tissue Total RNA Extraction Kit is stored dry at room temperature and can be stored for up to one year if it remains sealed. Buffers may form precipitates during storage. If this occurs, please warm the buffer to 37 until the precipitates are completely dissolved. 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 use. 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 use of this kit as described in this User s Guide, all potentially hazardous materials (i.e. materials that may have come in contact with genetically recombinant samples) including tubes, tips and materials should be processed and disposed of according to 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 experiments described in this User s Guide. Some applications that may be performed with this kit may infringe upon existing patents in certain countries. The purchase of this kit does not include or provide a license to perform patented applications. Users may be required to obtain a license depending on country and application. We do not condone nor recommend the unlicensed use of a patented application. 2

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 year from the date of purchase. If any issues are discovered relating to compromise in product quality, immediately contact BIONEER s Customer Service Center (order@bioneer.com). BIONEER does not assume liability for misuse of the product, i.e. usage of the product for any purposes other than its intended purpose as described in the User s Guide. BIONEER assumes liability under the condition that users disclose all information related to the problem to BIONEER in written form within 30 days of occurrence. VII. Technical Assistance At Bioneer, we pride ourselves on being responsive to your needs. 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: +82-42-930-8777 Email: sales@bioneer.com - In North America Tel: +1-877-264-4300 Email:support@bioneer.us.com VIII. Quality Management Every aspect of our quality management system from product development, production to quality assurance and supplier qualification meets the world-class standards. Each lot of MagListo 5M Tissue Total RNA Extraction Kit is carefully tested by the quality control team. 3

IX. Product 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. X. Principle The MagListo 5M Tissue Total RNA Extraction Kit is designed for the extraction of high-quality total RNA from cultured cells. Chemotropic agents in Binding Buffer contains guanidine hydrochloride and guanidine thiocyanate, which remove water molecules around RNA and silica-coated magnetic beads surface resulting in RNA captured by silica coated magnetic beads. The magnetic beads and RNA complexes are pulled and fixed on the tube wall using a magnetic force, followed by being washed with Washing Buffers to remove debris and excessive salts. The captured RNAs are then eluted by Elution Buffer, an aqueous solution with optimal ph. Sample Lysis Binding Washing Elution 4

XI. 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) 3. 1.5 ml or 2 ml tube (mini scale) 4. 50 ml tube (midi scale) 5. Vortex mixer 6. Absolute ethanol 7. Blow dryer or heat gun or dry oven 8. MagListo Magnetic Separation Rack Choice of the right Magnetic Separation Rack Tube MagListo Magnetic Separation Rack Cat.no 1.5 ml or 2 ml microcentrifuge tube MagListo -2 Magnetic Separation Rack TM-1010 50 ml centrifuge tube MagListo -50 Magnetic Separation Rack TM-1030 (Note) Please refer to the ordering information table on the latter part of the manual which contains the appropriate catalog number for specific size of tubes. 5

XII. Protocols Before you begin 1. MagListo 5M Tissue Total RNA Extraction Kit 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. Buffer 2 (1 st Washing) and Buffer 3 (2 nd Washing) are supplied as concentrated solutions. Before using for the first time, add absolute ethanol as indicated on the bottles. 4. 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). 5. How to use MagListo Magnetic Separation Rack. - Attachment Combine the magnet plate to the stand. Stand 1 Magnet plate - Discard solution Discard solution by inverting the MagListo rack. The silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discard solution, invert the rack completely for the solution not to smear on the rack. 2 - Detachment Push up the magnet plate gently. 3 6

RNA Extraction from Animal Tissue in Mini ( 20 mg of tissue) or Midi ( 200 mg of tissue) Scale Tissue Lysis & homogenization (Either Step 1 or Step 2 & 3) 1. Disruption and homogenization using a rotor-stator homogenizer : Place the weighed (fresh, frozen, or RNAlater -stabilized) tissue 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 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 thaw (go to step 3). 3. Add 400 µl (mini) / 4 ml (midi) of Buffer 1 (Binding) to the tissue sample, then completely homogenize the tissue by pipetting or vortexing for at least 1 min. (Note) Insufficient homogenization can decrease the RNA purification yield, and also cause clogging of Magnetic NanoBeads 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. For mini scale extraction, please transfer the lysate to a 2 ml (or 1.5 ml) tube. B. For midi scale extraction, please transfer the lysate to a 50 ml tube. RNA binding 4. Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to the tube and mix by vortexing or shaking. 5. Add 100 µl (mini) / 1 ml (midi) of Magnetic NanoBeads solution to the tube and mix by vortexing or shaking. (Note) Please vortex Magnetic NanoBeads solution well before use. 6. Place the tube in MagListo -2 (mini) / MagListo -50 (midi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to magnet. 7. Keeping the tubes in MagListo rack with the magnet attached, discard the supernatant and remove the remaining supernatant carefully using a paper towel by blotting. (Optional: If performing optional ONE Step RNA Clean up, follow steps (page 12) after performing this step.) 7

Washing 8. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of Buffer 2 (1 st Washing) to the tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 9. Place the tubes in MagListo rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 10. Keeping the tubes in the MagListo rack with the magnet attached, discard the supernatant and completely remove the remaining supernatant using a paper towel by blotting. 11. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of Buffer 3 (2 nd Washing) to the tube. Close the cap and mix by vortexing or vigorous shaking until the beads are fully resuspended. 12. Repeat the above steps 9 and 10 for additional washing. 13. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of absolute ethanol to the tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 14. Repeat the above steps 9 and 10 for additional washing. Drying 15. With a heat gun or a blow dryer, completely dry the beads with the tube open 3 cm away from the top of the tube. (mini: >1 min) (Note) Alternatively, the beads can be dried with a dry oven at 65 for current times. (mini: >5 min, midi: >15 min) Please use the clean bench during the drying procedure to prevent RNase or other aerosol contamination. Elution 16. Add >50 μl (mini) / >500 μl (midi) of Buffer 4 (Elution) to the tube with the magnet plate detached and resuspend RNA by vortexing or pipetting. 17. Incubate the tube at 55-65 for 2 min. Warm up the tube with a heat block, a heat gun or a blow dryer. 18. Vortex the tube for 15 sec. 19. Attach the magnet plate to MagListo rack and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 20. Keeping the tube in MagListo rack with the magnet attached, carefully take the supernatant containing RNA to a sterile microcentrifuge tube. 21. Discard the used Magnetic NanoBeads. Do not reuse the beads. 8

RNA Extraction from Cultured Cells in Mini ( 3 x 10 6 cells) or Midi ( 2 x 10 7 cells) Scale Harvest of cells 1. Cells grown in suspension: Count the cell number, then centrifuge the proper number of cells at 300 xg 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. 2a. 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 the RNA extraction) 2b. 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 xg for 5 min. Discard supernatant carefully and go to lysis & homogenization (go to step 3). Lysis & homogenization 3. Add 400 µl (mini) / 4 ml (midi) of Buffer 1 (Binding) to the cell culture dish or the collected cell pellet and completely homogenize the cells by pipetting or vortexing for at least 1 min. (Note) Insufficient homogenization can decrease the RNA purification yield, and also cause clogging of Magnetic NanoBeads 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. For mini scale extraction, please transfer the lysate to a 2 ml (or 1.5 ml) tube. B. For midi scale extraction, please transfer the lysate to a 50 ml tube. RNA binding 4. Add 200 µl (mini) / 2 ml (midi) of absolute ethanol to the tube and mix by vortexing or shaking. Add 100 µl (mini) / 1 ml (midi) of Magnetic NanoBeads solution to the tube and mix by vortexing or shaking. (Note) Please vortex Magnetic NanoBeads solution well before use. 5. Place the tube in MagListo -2 (mini) / MagListo -50 (midi) Magnetic Separation Rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to magnet. 6. Keeping the tubes from MagListo rack with the magnet attached, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. (Optional: If performing optional ONE Step RNA Cleanup, follow steps (page 12) after performing this step) 9

Washing 7. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of Buffer 2 (1 st Washing) to the tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 8. Place the tubes in MagListo rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 9. Keeping the tubes in the MagListo rack with the magnet attached, discard the supernatant and completely remove the remaining supernatant using a paper towel by blotting. 10. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of Buffer 3 (2 nd Washing) to the tube. Close the cap and mix by vortexing or vigorous shaking until the beads are fully resuspended. 11. Repeat the above step 9 and 10 for additional washing. 12. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of absolute ethanol to the tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 13. Repeat the above step 9 and 10 for additional washing. Drying 14. With a heat gun or a blow dryer, completely dry the beads with the tube open 3 cm away from the top of the tube. (mini: >1 min) (Note) Alternatively, the beads can be dried with a dry oven at 65 for current times. (mini: >5 min, midi: >15 min) Please use the clean bench during the drying procedure to prevent RNase or other aerosol contamination. Elution 15. Add >50 μl (mini) / >500 μl (midi) of Buffer 4 (Elution) to the tube with the magnet plate detached and resuspend by vortexing or pipetting. 16. Incubate the tube at 55-65 for 2 min. Warm up the tube with a heat block, a heat gun or a blow dryer. 17. Vortex the tube for 15 sec. 18. Attach the magnet plate to MagListo rack and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 19. Keeping the tube in MagListo rack with the magnet attached, carefully take the supernatant containing RNA to a sterile microcentrifuge tube. 20. Discard the used Magnetic NanoBeads. Do not reuse the beads. 10

Summary of reagent volumes required in each step of Tissue Total RNA Extraction. Step Buffer Mini scale ( 20 mg tissue) Midi scale ( 200 mg tissue) Tissue Lysis Buffer 1 (Binding) 400 μl 4 ml Addition of Ethanol Absolute ethanol 200 μl 2 ml RNA Binding Magnetic NanoBeads - RNA 100 μl 1 ml 1 st Washing Buffer 2 (1 st Washing) 800 μl 8 ml 2 nd Washing Buffer 3 (2 nd Washing) 800 μl 8 ml 3 rd Washing Absolute ethanol 800 μl 8 ml Elution Buffer 4 (Elution) >50 μl >500 μl Summary of reagent volumes required in each step of Cell Total RNA Extraction Step Buffer Mini scale ( 3 x 10 6 cells) Midi scale ( 2 x 10 7 cells) Cell Lysis Buffer 1 (Binding) 400 μl 4 ml Addition of Ethanol Absolute ethanol 200 μl 2 ml RNA Binding Magnetic NanoBeads - RNA 100 μl 1 ml 1 st Washing Buffer 2 (1 st Washing) 800 μl 8 ml 2 nd Washing Buffer 3 (2 nd Washing) 800 μl 8 ml 3 rd Washing Absolute ethanol 800 μl 8 ml Elution Buffer 4 (Elution) >50 μl >500 μl 11

ONE-Step RNA Clean Up Protocol This protocol is to remove DNA for use in certain applications. 1. Detach the magnet plate from MagListo rack. Add 800 µl (mini) / 8 ml (midi) of absolute ethanol to the tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 2. Place the tube in MagListo -2 (mini) / MagListo -50 (midi) with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 3. Keeping the tube from MagListo rack with the magnet attached, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. 4. With a heat gun or a blow dryer, completely dry the beads with the tube open 3 cm away from the top of the tube. (mini: >2 min, midi: >5 min) (Note) Alternatively, the beads can be dried with a dry oven at 65 for current times. (mini: > 5 min, midi: >15 min) Please use the clean bench during the drying procedure to prevent RNase or other aerosol contamination. 5. Add DNAse Reaction Buffer and RNase-Free DNase to the tube. (mini: up to 70 µl, midi: up to 700 µl) 6. Detach the magnet plate from MagListo rack and Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 7. Place on the benchtop (20 30 C) for 20 min. 8. 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 tube. Close the cap and mix by vortexing or shaking until the beads are fully resuspended. 9. Place the tube in MagListo rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 10. Keeping the tube in MagListo rack with the magnet attached, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. 11. Go to 2 nd Washing step 11 (Page 8, 10) and follow the instruction described above for the remaining steps. 12

RNA Clean up Protocol This protocol is to remove enzymes, buffers, or chemical inhibitors and concentrate RNA for use in certain applications. 1. Transfer RNase-free water into RNA sample to a volume of 100 µl. 2. Add 100 µl of Buffer 1 (Binding) and mix thoroughly. 3. Add 200 µl of absolute ethanol and mix well. 4. Add 100 µl of Magnetic NanoBeads solution to the tube and mix by vortexing or shaking (Note) Please vortex Magnetic NanoBeads solution well before use. 5. Place the tube in MagListo rack with the magnet plate attached and invert the tube 3-4 times gently until the beads tightly bind to the magnet. 6. Keeping the tube in MagListo rack with the magnet attached, discard the supernatant and remove the remaining supernatant using a paper towel by blotting. 7. Go to 1 st Washing step 8 (Page 8, 10) and follow the instruction described above for the remaining steps. 13

XIII. Troubleshooting Guide Comments and suggestions Insufficient homogenization: (Refer to Page 7) Add Buffer 1 (Binding) to the tissue (cells) and completely homogenize the tissue (cells) by vortexing for at least 1 min or increase the vortexing time for the sufficient homogenization up to 5 to 10 minutes. Use of an excess amount of sample: Use the proper amount of samples according to the instructions of this User s Guide. Low or no yield Insufficient beads dried: Beads must be completely dried. Any leftover of ethanol can decrease the RNA purification yield. Insufficient incubation during the Elution step: RNA could remain attached to the beads if the incubation time is not enough. To prevent this, please repeat the elution process with the increased incubation time. Incomplete removal of cell culture medium: 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. Aggregation of Magnetic NanoBeads RNA degradation Inhibition of downstream enzymatic reactions Low A 260/280 ratio Insufficient homogenization: (Refer to Page 7) Add Buffer 1 (Binding) to the tissue (cells) and completely homogenize the tissue (cells) by vortexing for at least 1min or increase the vortexing time for the sufficient homogenization. Use of an excess amount of sample: Use the proper amount of samples according to the instructions of this User s Guide. RNase contamination: 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. Inappropriate handling of harvested samples: If the extraction is not performed right after harvesting the samples, store the samples at -80 or lysis the samples with Buffer 1 (Binding) and then store at -80. Insufficient beads dried: Beads must be completely dried. Any leftover in the ethanol can decrease the RNA purification yield. Insufficient washing during the beads suspension process: Insufficient suspension of beads during the washing step causes salts to remain in the purified RNA. Recommendation is to suspend the beads thoroughly during the washing process. Insufficient beads dried: Beads must be completely dried. Any leftover in the ethanol can decrease the RNA purification yield. Insufficient washing during the beads suspension process: Insufficient suspension of beads during the washing step causes salts to remain in the purified RNA. Recommendation is to suspend the beads thoroughly during the washing process. 14

XIV. Ordering Information Cat no. Product Description Size K-3601SM/ K-3601/ K-3600 K-3602/ K-3603 K-3604/ K-3605 K-3606/ K-3607 K-3608/ K-3609 K-3610/ K-3611 K-3612/ K-3613 K-3614/ K-3615 MagListo 5M Plasmid Extraction Kit MagListo 5M Genomic DNA Extraction Kit MagListo 5M Plant Genomic DNA Extraction Kit MagListo 5M Gel Extraction Kit MagListo 5M PCR Purification Kit MagListo 5M Cell Total RNA Extraction Kit MagListo 5M Tissue Total RNA Extraction Kit MagListo 5M Forensic Sample DNA Extraction Kit 8 rxn / 100 rxn/ 500 rxn (mini*) 8 rxn / 100 rxn (mini*) 8 rxn / 100 rxn (mini*) 8 rxn / 100 rxn (mini*) 8 rxn / 100 rxn (mini*) 8 rxn /100 rxn (mini*) 8 rxn /100 rxn (mini*) 8 rxn /100 rxn (mini) TM-1000 MagListo -8Ch Magnetic Separation Rack 1 ml tube x 8 holes TM-1010 MagListo -2 Magnetic Separation Rack 2 ml tube x 8 holes TM-1020 MagListo -15 Magnetic Separation Rack 15 ml tube x 6 holes TM-1030 MagListo -50 Magnetic Separation Rack 50 ml tube x 3 holes TM-1040 MagListo -96 Magnetic Separation Rack 96 well plate x 1 ea TM-1100 MagListo Magnetic Separation Rack Bundle Set Each of MagListo TM -2, -15, -50, and -96 (4 racks) K-3601-A Blow Dryer 1 ea HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk HT-20-NG 2 ml microcentrifuge tube 500 ea / pk 91081 15 ml tube 500 ea / pk 91082 50 ml tube 500 ea / pk 91083 1 ml tube with 8-cap strip 100 ea / pk 15

XV. Explanation Symbols Catalog Number Contains sufficient for (n) tests USE BY Consult Instruction For Use Caution, Batch code consult accompanyi ng Temperatu re Limitation Research Use Only documents Manufactur er Caution, Potential Biohazard DO NOT REUSE 16

[Note] 17

8-11 Munpyeongseo-ro, Daedeok-gu, Daejeon, 34302, Republic of Korea +82-42-930-8777 (Korea: 1588-9788) +82-42-930-8688 sales@bioneer.com 1301 Marina Village PKWY, Suite 110, Alameda, CA 94501, USA +1-877-264-4300 (Toll-free) +1-510-865-0350 order.usa@bioneer.com us.bioneer.com Korea Bio Park BLDG #B-702, 700 Daewangpangyo-ro, Bundang-gu, Seongnam-si Gyeonggi-do, 13488, Republic of Korea +82-31-628-0500 +82-31-628-0555 sales@bioneer.co.kr www.bioneer.co.kr 18