Kit for the extraction of genomic DNA from blood and tissue using MagListo Version No.: 2.0 (2017-03) 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: order@bioneer.co.kr MagListo is a trademark of Bioneer Corporation. Copyright 2017. Bioneer Corporation. All Rights Reserved.
Contents I. Overview II. Kit components III. Storage IV. Intended use V. Safety warnings and precautions VI. Warranty and liability VII. Technical assistance VIII. Quality management IX. Kit specifications Extraction of genomic DNA small amount of sample Recommended amounts of starting sample X. Sample preparation XI. Principle 1 1 2 2 2 2 3 3 4 4 4 5 6 XII. Magnetic Nano Bead Information 6 XIII. XIV. XV. XVI. Guidelines for MagListo Magnetic Separation Rack Materials and Equipment Needed But Not Provided Types of the Magnetic Separation Rack Procedure Protocols 7 8 8 9 10 Before You Begin 10 A. DNA Extraction from Whole Blood 10 B. DNA Extraction from Cultured Cell 15
C. DNA Extraction from Animal Tissue 20 D. DNA Extraction from Bacterial cells (Gram-Negative Bacteria) 25 E. DNA Extraction from Bacterial cells (Gram-Positive Bacteria) 26 F. DNA Clean-Up (DNA Purification) 27 XVII. Appendix Troubleshooting guide 29 29 Experimental data 32 XVIII. Ordering information XIX. Explanation symbols 35 36
I. Overview Description MagListo 5M Genomic DNA Extraction Kit utilizes Magnetic Nano Beads to extract total DNA from various of sources, such as whole blood, animal tissue and 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 Gene Cloning, PCR, Real-Time PCR, Southern Blotting, SNP genotyping II. Kit Components MagListo 5M Genomic DNA Extraction Kit *K-3603 Buffer 1 (Lysis) Buffer 2 (Binding) Buffer 3 (1 st Washing) Buffer 4 (2 nd Washing) Buffer 5 (3 rd Washing) Buffer 6 (Elution) Magnetic Nano Bead - DNA Proteinase K powder, lyophilized RNase A powder, lyophilized 25 ml x 1 ea 25 ml x 1 ea 120 ml x 1 ea 80 ml x 1 ea 120ml x 1 ea 25 ml x 1 ea 1.8 ml x 6 ea 25 mg x 2 ea 24 mg x 2 ea *Mini 100 rxn, Midi 15 rxn, Maxi 8 rxn 1
III. Storage MagListo 5M Genomic DNA 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 Genomic DNA 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 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 liability for misuse of the product, i.e. using the product for any purposes 2
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: +82-42-930-8777 Email: sales@bioneer.co.kr 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 to supplier qualification ensures that our products meet the world-class standards. Each lot of MagListo 5M Genomic DNA Extraction Kit is carefully tested by the quality control team. 3
IX. Kit Specifications Sample Type Mini (Typical yield) Midi (Typical yield) Maxi (Typical yield) Whole blood 200 μl ( < 10 μg) 2 ml ( < 80 μg) 4 ml ( < 150 μg) Cultured cell ~ 1 x 10 6 ( < 12 μg) ~ 5 x 10 6 (< 60 μg) ~ 1 x 10 7 (< 120 μg) Animal tissue ~ 25 mg ( < 10 μg) ~ 100 mg ( < 40 μg) ~ 250 mg (< 120 μg) Bacteria (Gram (-), (+)) ~ 1 x 10 9 (< 15 μg) ~ 5 x 10 9 (< 80 μg) ~ 1 x 10 10 (< 150 μg) Expected purity A 260 / 280 > 1.8 * The DNA yield from samples with a low number of cells may be less than the figures shown in the table. ** For cultured cells, samples with cell number of ~10 4 can be used for micro scale extraction. Extraction of genomic DNA from small amount of sample MagListo 5M Genomic DNA Extraction Kit is able to extract genomic DNA from sample of low quantity. If sample contains low number of cells (< 1x10 4 ) or has small amount of DNA, it is recommended to add about 4 μg of carrier RNA to the starting sample. Ensure that the carrier DNA does not interfere with your downstream application. Carrier RNA can be removed later by RNase digestion. Please see DNA Extraction from Cultured Cell for Micro in page 15 for more details about DNA extraction from samples with a low number of cells. Recommended amounts 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 9.6 1.2 x 10 6 12 well 4 4 x 10 5 24 well 2 2 x 10 5 48 well 1 1 x 10 5 96 well 0.35-0.6 4 x 10 4 Dishes 4
35 mm 8 1.2 x 10 6 60 mm 21 3 x 10 6 100 mm 55 8 x 10 6 150 mm 148 2 x 10 7 Flasks 50 ml 25 2.5 x 10 6 300 ml 75 1 x 10 7 X. Sample preparation Several factors, such as harvesting method and storage of starting samples can influence the yield and purity of DNA. All specimens must be stored in a freezer or used immediately after collection. It is recommended to process the sample as soon as possible on ice, and to avoid repeated freezing and thawing. Blood Blood sample should immediately be used or stored in a collection tube containing the anticoagulants for blood (EDTA or ACDs). Samples can be stored for few days at 4 and at up to 1 year at -70. It is recommended rapidly thaw frozen blood sample in water bath (37 ) and to keep the blood sample on ice until use. Cultured cells Cultured cells can easily be harvested using a centrifuge. However, it might be difficult to extract genomic DNA if cultured cells are too clustered. In this case, trypsin can be used to detach each cell from the cluster. For extraction using MagListo 5M Genomic DNA Extraction Kit, number of cells should be less than 1 x 10 7, which is calculated with a cell counter. It is recommended to keep samples on ice until use. Tissue Tissue samples should immediately be used or stored at -70 upon harvest 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. Bacterial cells Bacterial cells can be processed in a shaking incubator for 12-16 hours at 37. Optimal results can be obtained when harvested bacterial cells are immediately used or stored at between -20 and -80. Additional bacteriolytic agents like lysozome or lysostaphin should be used to break the multilayered cell wall of Gram-positive bacteria. For gram negative bacteria, these agents are not needed. 5
XI. Principle MagListo 5M Genomic DNA Extraction Kit is designed for the extraction of genomic DNA including high molecular weight DNA (up to 40 kb). The overall principle is based on the adsorption of DNA onto the Magnetic Nano Bead by chaotropic salt. For example, chaotropic agents in Buffer 2 (Binding) contains guanidine hydrochloride, as which removes water molecules around DNA and silica coated magnetic beads surface resulting in genomic DNA then being captured by magnetic beads. The Magnetic Nano Bead and nucleic acid 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 nucleic acids are then eluted by Buffer 6 (Elution), an aqueous solution with an optimal ph. Sample Lysis Binding Washing Elution 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 DNA and With the Magnetic Nano Beads are then isolated using external magnetic field. Features Fast binding guarantees higher throughput automation Large surface area enables better sensitive assay Globular structure increases specificity by decreasing non-specific binding 6
Specification Matrix Silica-coated Fe 3 O 4 AccuNanoBead 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 TM Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic NanoBeads. Bioneer offers various racks of different sizes- MagListo -8Ch for 8-tube strip and multipipette, MagListo -2 for 2 ml tube, MagListo -15 for 15 ml tube, MagListo -50 for 50 ml tube and MagListo -96 for 96 well microplate. 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 breaks, do not discard it with bare hands as the sharp edges may cause injury. 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 do not spill 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. 7
XIV. Materials and Equipment Needed But Not Provided 1. 1.5 ml or 2 ml tube, 15 ml tube, 50 ml tube 2. Vortex mixer 3. Absolute ethanol 4. Thermal block or dry oven 5. Phosphate buffered saline (PBS) 6. MagListo Magnetic Separation Rack Types of the Magnetic Separation Rack Tube 1 ml tube with 8-cap strip 1.5 ml or 2 ml microcentrifuge tube 15 ml tube 50 ml tube MagListo Magnetic Separation Rack MagListo -8Ch Magnetic Separation Rack (Cat. no. TM-1000) MagListo -2 Magnetic Separation Rack (Cat. no. TM-1010) MagListo -15 Magnetic Separation Rack (Cat. no. TM-1020) MagListo -50 Magnetic Separation Rack (Cat. no. 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. 8
XV. Procedure-Genomic DNA Extraction 9
XVI. Protocols Before you begin 1. Completely dissolve Proteinase K (KB-0111) in 1,250 ul of nuclease-free water. Dissolved Proteinase K should be stored at 4. 2. Completely dissolve RNase A (KB-3101) in 600 ul of nuclease-free water. Dissolved RNase A should be stored at 4. 3. Buffer 2 (Binding) and Buffer 3 (1 st Washing) contain chaotropic salt. You should take appropriate laboratory safety precautions and wear gloves when handling this buffer. 4. If there is any precipitate in Buffer 1 (Lysis), incubate on a heating block at 56. A. DNA Extraction from Whole Blood for Mini /Midi /Maxi scale 1. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see Before you begin ) to each tube. a. (Mini) Add Proteinase K to a 1.5 ml or 2 ml tube. b. (Midi) Add Proteinase K to a 15 ml tube. c. (Maxi) Add Proteinase K to a 50 ml tube. 2. Transfer 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of whole blood and buffy coat to the tubes containing proteinase K. (Note) If the sample volume is less than the indicated volume above, bring the sample volume to a total volume of 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) by adding 1X PBS to achieve maximum lysis efficiency and yield. 3. (Lysis: 3-4) Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer 2 (Binding) to each tube and mix thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to achieve maximum lysis efficiency. 4. Incubate the tubes at 60 for 10 min. 10
5. (DNA Precipitation) Add 400 μl (mini) / 4 ml (midi) / 8 ml (maxi) of absolute ethanol to each tube and mix well using a vortex mixer or by pipetting. 6. (DNA binding with Magnetic Nano Bead: 6-8) Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Magnetic Nano Bead 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. 7. Place the tube on the MagListo -2 (mini) / MagListo -15 (midi) / MagListo -50 (maxi) 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. 8. 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. During this process, the magnetic crude pellet remains attached to the side of tube. - 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. 11
9. (1 st washing: 9-12) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 500 μl (mini) / 3 ml (midi) / 5 ml (maxi) of Buffer 3 (1 st Washing) to the each tube and close the cap. Mix using a vortex mixer until the beads are fully resuspended. - Detachment of the magnet plate Detach the magnet plate gently by pulling it upwards. 10. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 11. 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. 12. Repeat the steps 9-11 to remove remaining pigments and other debris. 13. (2 nd washing) Repeat the steps 9-11 by adding 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer 4 (2 nd Washing) instead of Buffer 3 for additional washing. 14. (3 rd washing) Without removing the tubes from the MagListo Magnetic Separation Rack, add 700 μl (micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer 5 (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 5 onto the bead pellet, vortexing and/or vigorous shaking of the tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected. 15. Discard the supernatant and completely remove the remaining supernatant by blotting action. 12
- Add Buffer 5 and discard the supernatant 16. (Elution: 16-21) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Buffer 6 (Elution) or distilled water to the each tube and resuspend the bead pellet completely by pipetting or using a vortex mixer for 15 sec. 17. Incubate the tubes at 60 for 1 min. 18. Vortex the tubes for 15 sec. 19. 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. 20. Without removing the tube from the MagListo Magnetic Separation Rack, transfer supernatant containing DNA carefully to a new sterile microcentrifuge tube. 21. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads. 13
Summary of Reagents Volume in Each Step of DNA Extraction from Whole Blood Step Buffer Mini Midi Maxi Page Blood (+ PBS) 200 μl 2 ml 4 ml P. 10 Lysis Buffer 2 (Binding) 200 μl 2 ml 4 ml P. 10 DNA Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 10 Bead Binding Magnetic Nano Bead - DNA 100 μl 500 μl 1 ml P. 11 1 st Washing (Repeat) Buffer 3 (1 st Washing) 500 μl 3 ml 6 ml P. 12 2 nd Washing Buffer 4 (2 nd Washing) 700 μl 5 ml 10 ml P. 12 3 rd Washing Buffer 5 (3 rd Washing) 700 μl 8 ml 15 ml P. 12 Elution Buffer 6 (Elution) 100 μl 500 μl 1 ml P. 13 14
B. DNA Extraction from Cultured Cells for Micro /Mini /Midi /Maxi scale 1. Centrifuge the cultured cells (~ 1x10 4 (micro) / ~ 1x10 6 (mini) / ~ 5x10 6 (midi) / ~ 1x10 7 (maxi)) for 10 min at 300 x g. Discard the supernatant carefully without disturbing the pellet. (Note) For cultured cells with lower cell number than 1x10 4, micro scale as described in this protocol. 2. Resuspend the pellet in 100 μl (micro) / 200 μl (mini) / 1 ml (midi, maxi) of 1X PBS and transfer the resuspended cells to each tube. a. (Micro / Mini) Transfer the resuspended cells to a new 1.5 ml or 2 ml tube. b. (Midi / Maxi) Transfer the resuspended cells to a new 15 ml tube. 3. Add 10 μl (micro) / 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see Before you begin ) to the each tube. 4. If you require RNA-free genomic DNA is required, add up to 2 μl (micro) / 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A (see Before you begin ) to each tube and incubate the tubes for 5 min at room temperature. 5. (Lysis: 5-6) Add 100 μl (micro) / 200 μl (mini) / 1 ml (midi, maxi) of Buffer 2 (Binding) to the each tube and mix thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to achieve maximum lysis efficiency. 6. Incubate the tubes at 60 for 10min. 7. (DNA precipitation) Add 200 μl (micro) / 400 μl (mini) / 2 ml (midi, maxi) of absolute ethanol to each tube and mix well using a vortex mixer or by pipetting. 8. (DNA binding with Magnetic Nano Bead: 8-10) Add 100 μl (micro, mini) / 500 μl (midi) / 1 ml (maxi) of Magnetic Nano Bead solution to each tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. 15
(Note) Magnetic Nano Bead Solution contains Magnetic Nano Beads. Please shake well or mix with a vortex mixer before use. 9. Place the tubes on the MagListo -2 (micro, mini) / MagListo -15 (midi, maxi) 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. 10. 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. - 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 spill on the rack. 11. (1 st washing: 11-13) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 700 μl (micro, mini) / 5 ml (midi) / 10 ml (maxi) of Buffer 3 (1 st Washing) to each tube and close the cap. Mix using a vortex mixer until the beads are fully resuspended. 16
- Detachment of the magnet plate Detach the magnet plate gently by pulling it upwards. 12. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 13. 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. 14. (2 nd washing) Repeat the steps 11-13 by adding 700 μl (micro, mini) / 5 ml (midi) / 10 ml (maxi) Buffer 4 (2 nd Washing) instead of Buffer 3 for additional washing. 15. (3 rd washing) Without removing the tubes from the MagListo Magnetic Separation Rack, add 700 μl (micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer 5 (3 rd Washing) to the opposite side of the bead pellet. Close the cap and gently invert the rack twice in order to remove ethanol from the sample. (Note) Direct pipetting of Buffer 5 onto the bead pellet, vortexing and/or vigorous shaking of the tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected. 16. Discard the supernatant and completely remover the remaining supernatant by blotting action. - Add Buffer 5 and discard the supernatant 17
17. (Elution: 17-21) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 50 μl (micro) / 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Buffer 6 (Elution) or distilled water to the each tube completely resuspend the bead pellet by pipetting or using a vortex mixer for 15 sec. 18. Incubate the tubes at 60 for 1 min. 19. Place the tubes on the MagListo Magnetic Separation Rack, attach the magnet plate and invert the track gently 3 to 4 times until the beads bind tightly to the magnet. 20. Without removing the tubes from the MagListo Magnetic Separation Rack, transfer supernatant containing DNA to a new sterile microcentrifuge tube. 21. Discard the tubes with remaining Magnetic Nano Bead pellet. Do not reuse the beads. 18
Summary of Reagents Volume in Each Step of DNA Extraction from Cultured Cell Step Buffer Micro Mini Midi Maxi Page Cultured Cell ~ 1 x 10 4 ~ 1 x 10 6 ~ 5 x 10 6 ~1 x 10 7 P. 15 Lysis Buffer 2 (Binding) 100 μl 200 μl 1 ml 1 ml P. 15 DNA Precipitation Absolute Ethanol 200 μl 400 μl 2 ml 2 ml P. 15 Bead Binding Magnetic Nano Bead - DNA 100 μl 100 μl 500 μl 1 ml P. 15 1 st Washing Buffer 3 (1 st Washing) 700 μl 700 μl 5 ml 10 ml P. 16 2 nd Washing Buffer 4 (2 nd Washing) 700 μl 700 μl 5 ml 10 ml P. 17 3 rd Washing Buffer 5 (3 rd Washing) 700 μl 700 μl 8 ml 15ml P. 17 Elution Buffer 6 (Elution) 50 μl 100 μl 500 μl 1 ml P. 18 19
C. DNA Extraction from Animal Tissue for Mini /Midi /Maxi scale 1. (Homogenization) Disrupt (or homogenize) the tissue sample (~ 25 mg (mini) / ~ 100 mg (midi) / ~ 250 mg (maxi)) with a mortar and a pestle. Transfer disrupted or homogenized sample to each tube. a. (Mini) Transfer the homogenized tissue sample to a 1.5 ml or 2 ml tube. b. (Midi) Transfer the homogenized tissue sample to a 15 ml tube. c. (Maxi) Transfer the homogenized tissue sample to a 50 ml tube. (Note) Immediately place the weighted, fresh or frozen tissue in liquid nitrogen and grind to a fine powder with a mortar and a pestle. Final yield of DNA will depend on the amount and the type of the tissue used. 2. (Lysis: 2-6) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Buffer l (Lysis) to each tube. 3. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see Before you begin ) to the each tube and mix thoroughly using a vortex mixer. 4. If you require RNA-free genomic DNA is required, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A (see Before you begin ) and incubate the tubes for 2 min at room temperature. 5. Incubate the tubes at 60 until the tissue is completely lysed. 6. Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer 2 (Binding) to the each tube and mix thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to achieve maximum lysis efficiency. 7. (DNA precipitation) Add 400 μl (mini) / 4 ml (midi) / 8 ml (maxi) of absolute ethanol to each tube and mix well using a vortex mixer or by pipetting. 8. (DNA binding with Magnetic Nano Bead: 8-10) Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Magnetic Nano Bead solution to the each tube and mix thoroughly using a vortex mixer until the beads are fully resuspended. 20
(Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix using a vortex mixer before use. 9. Place the tubes on the MagListo -2 (mini) / MagListo -15 (midi) / MagListo -50 (maxi) Magnetic Separation Rack with the magnet plate 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. 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. - 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 spill on the rack. 11. (1 st washing: 11-13) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer 3 (1 st Washing) to each tube and close the cap. Mix using a vortex mixer until the beads are fully resuspended. 21
- Detachment of the magnet plate Detach the magnet plate gently by pulling it upwards. 12. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 13. 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. 14. (2 nd washing) Repeat the steps 11-13 by adding 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) Buffer 4 (2 nd Washing) instead of Buffer 3 for additional washing. 15. (3 rd washing) Without removing the tubes from MagListo Magnetic Separation Rack, add 700 μl (micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer 5 (3 rd Washing) to the opposite side of the bead pellet. Close the cap and invert the rack twice in order to remove ethanol from the sample. (Note) Direct pipetting of Buffer 5 onto the bead pellet, vortexing or vigorous shaking of the tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected. 16. Discard the supernatant and completely remove the remaining supernatant by blotting action. - Add Buffer 5 and discard the supernatant 22
17. (Elution: 17-21) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Buffer 6 (Elution) or distilled water to each tube and completely resuspend the bead pellet by pipetting or using a vortex mixer for 15 sec. 18. Incubate the tubes at 60 for 1 min. 19. 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. 20. Without removing the tubes from MagListo Magnetic Separation Rack, transfer supernatant containing DNA carefully to a new sterile microcentrifuge tube. 21. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads. 23
Summary of Reagents Volume in Each Step of DNA Extraction from Animal Tissue Step Buffer Mini Midi Maxi Page Animal tissue ~ 25 mg ~ 100 mg ~ 250 mg P. 20 Lysis Buffer l (Lysis) 180 μl 1.8 ml 3.6 ml P. 20 Buffer 2 (Binding) 200 μl 2 ml 4 ml P. 20 DNA Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 20 Bead Binding Magnetic Nano Bead DNA 100 μl 500 μl 1 ml P. 20 1 st Washing Buffer 3 (1 st Washing) 700 μl 5 ml 10 ml P. 21 2 nd Washing Buffer 4 (2 nd Washing) 700 μl 5 ml 10 ml P. 22 3 rd Washing Buffer 5 (3 rd Washing) 700 μl 8 ml 15 ml P. 22 Elution Buffer 6 (Elution) 100 μl 500 μl 1 ml P. 23 24
D. DNA Extraction from Bacterial Cells (Gram-Negative Bacteria) for Mini/Midi/Maxi 1. (Cell collection) Collect the bacterial cells (~1x10 9 (mini) / ~5x10 9 (midi) / ~1x10 10 (maxi)) by centrifuging at 6000 x g (> 8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min). Discard the supernatant (media) by using a pipette. 2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Buffer l (Lysis) to the collected cell pellet and completely resuspend using a vortex mixer or by pipetting. 3. Transfer the cell suspension to each tube. a. (Mini) Transfer the cell suspension to a 1.5 ml or 2 ml tube. b. (Midi) Transfer the cell suspension to a 15 ml tube. c. (Maxi) Transfer the cell suspension to a 50 ml tube. 4. Go to step 3 of C. DNA Extraction from Animal Tissue in page 20 and follow the instructions accordingly. 25
E. DNA Extraction from Bacterial Cells (Gram-Positive Bacteria) for Mini/Midi/Maxi 1. (Cell collection) Collect the bacterial cells (~ 1x10 9 (mini) / ~ 5x10 9 (midi) / ~ 1x10 10 (maxi)) by centrifuging at 6,000 x g (>8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min). Discard the supernatant (media) by using a pipette. 2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Lysis Buffer for Gram-Positive bacteria (not provided) to the collected cell pellet and completely resuspend using a vortex mixer or by pipetting. (Note) Lysis Buffer for Gram-Positive bacteria can be prepared by using this formulation: 20 mm Tris- HCl (ph8.0), 2 mm sodium EDTA and 1.2% Triton X-100 3. Transfer the cell suspension to each tube. a. (Mini) Transfer the cell suspension to a 1.5 ml or 2 ml tube. b. (Midi) Transfer the cell suspension to a 15 ml tube. c. (Maxi) Transfer the cell suspension to a 50 ml tube. 4. (Lysis: 4-9) Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of lysozyme (100 mg/ml, not provided) to each tube and mix thoroughly using a vortex mixer. 5. If you require RNA-free genomic DNA, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A (see Before you begin ). 6. Incubate the tubes at 37 for 30 min. 7. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see Before you begin ) to each tube. 8. Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer 2 (Binding) to each tube and mix thoroughly using a vortex mixer. 9. Incubate the tubes at 60 for 30 min or until bacterial cells are completely lysed. 10. Go to step 7 of C. DNA Extraction from Animal Tissue in page 20 and follow the instructions accordingly. 26
F. DNA Clean-Up (DNA Purification) 1. Transfer the eluted DNA or enzyme reaction product to a new sterile tube. a. (Mini) Transfer the eluate to a 1.5 ml or 2 ml tube b. (Midi / Maxi) Transfer the eluate to a 15 ml tube 2. If you require RNA-free genomic DNA, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A (see Before you begin ) and incubate the tubes for 2 min at room temperature. 3. (Binding) Add 1 volume of Buffer 2 (Binding) to 1 volume of the eluted DNA and mix completely using a vortex mixer. 4. (DNA precipitation) Add 3 volumes of absolute ethanol to 1 volume of the eluted DNA and mix well using a vortex mixer. 5. (DNA binding with Magnetic Nano Bead: 5-7) Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Magnetic Nano Bead solution to 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. 6. Place the tubes on the MagListo -2 (mini) / MagListo -15 (midi, maxi) 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. 27
7. 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. - 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 spill on the rack. 8. (1 st washing: 8-10) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer 4 (2 nd Washing) to 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 magnet. 10. Without removing the tubes from MagListo Magnetic Separation Rack, discard the supernatant and remove the remaining supernatant on a paper towel by blotting action. 11. Go to step 15 of C. DNA Extraction from Animal Tissue in page 22 and follow the instructions accordingly. 28
XVII. Appendix Troubleshooting guide If you have problems during genomic DNA extraction, please use the Troubleshooting Guide. This troubleshooting guide will help you to solve problem that may arise during DNA 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. Cells may not have been lysed properly, especially in the case of tissue cells. Please, ensure that the turbid sample changes to a clear solution, indicating that the protein digestion has occurred. Extend the incubation time if tissue cells are still not lysed. The amount of time for complete lysis varies depending on the Low yield of DNA type of tissue or sample type used. If a cell mass still remains after the overnight incubation, centrifuge the sample and use supernatant for DNA extraction. A shaking water bath should be used for efficient cell lysis. 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 genomic DNA. Elution may have been incomplete. Please extend incubation time up to 3 minutes at elution step to improve the DNA. In addition, make sure that Magnetic Nano Beads are suspended completely in the eluting solution during incubation. 29
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. Shake or mix with a vortex mixer sufficiently during incubation step. Beads may have been washed insufficiently. You must properly wash the beads in the 3 rd washing step. Remained ethanol can decrease the purity of DNA. Take Low A 260/280 ratio enough time to properly wash the beads. Incomplete suspension of beads during the washing step causes salts to remain in the purified DNA. Make sure that the beads are suspended thoroughly during the washing process. RNA may be present in the eluted DNA when both DNA and RNA are resent in Presence of RNA in the eluted DNA the sample. If you require RNA-free genomic DNA, add RNase A should be added to the sample before addition of Buffer 2 (Binding). If you wish to RNA from the eluted DNA, please refer to F. DNA Clean-Up (DNA Purification) protocol in page 20 for details. Excessively clustered Magnetic Nano Bead Excess amount of starting sample is used to extract DNA. Appropriate amount of starting material (see Kit Specification in page 4) should be used for efficient extraction of genomic DNA. Presence of a white precipitate in buffers A white precipitate may form in Buffer l (Lysis) or Buffer 2 (Binding) due to prolonged storage at low temperatures. Incubate Buffer l (Lysis) or Buffer 2 (Binding) at 60 to dissolve any precipitate in the buffer. 30
The DNA from old or incorrectly stored sample may often be degraded. As the DNA yield is highly dependent on storage conditions of samples, please use fresh samples for optimal results. In case of using stored tissue sample, it is Degraded DNA recommended to use sample stored at -70. Frequent freezing and thawing may result in lower DNA yield than expected. Avoid repeated freezing and thawing. Flotation of extracted DNA when loaded on an agarose gel Floating of DNA on an agarose gel is caused by the remaining ethanol in the eluted DNA. Ensure that the 3 rd Washing (ethanol removing) step in the protocol is properly performed. Remaining ethanol may also interrupt the enzymatic reaction. 31
Experimental data Figure1. Comparison of extracted genomic DNA from human whole blood (200μl) purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) M 1 2 3 4 5 6 7 M: Bioneer 1kb ladder 1-4: Extracted blood genomic DNA purified with Bioneer MagListo 5M Genomic DNA Extraction Kit 5-7: Extracted blood genomic DNA purified with competitor Q kit Sample Total Yield (ug) A260/A280 A260/A230 1 6.5 1.83 2.06 2 7.6 1.85 1.9 3 6 1.86 2.05 4 5.7 1.84 2.03 5 5.3 1.88 1.53 6 5.5 1.82 1.29 7 5.8 1.84 1.75 Figure2. Comparison of extracted genomic DNA from HeLa cell (1x10 6 ) purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) M 1 2 3 4 5 6 M: Bioneer 1kb ladder 1-4: Extracted cell genomic DNA purified with Bioneer MagListo 5M Genomic DNA Extraction Kit 5-6: Extracted cell genomic DNA purified with competitor Q kit Sample Total Yield (ug) A260/A280 A260/A230 1 23.5 1.94 1.97 2 24.4 1.95 1.97 3 21.3 1.89 2.06 4 27.2 1.88 2.04 5 21.5 1.99 2.42 6 25.1 2 2.41 32
Figure3. Comparison of extracted genomic DNA from bovine skeletal muscle tissue (15mg) purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) M 1 2 3 4 5 6 M: Bioneer 1kb ladder 1-4: Extracted tissue genomic DNA purified with Bioneer MagListo 5M Genomic DNA Extraction Kit 5-6: Extracted tissue genomic DNA purified with competitor Q kit Sample Total Yield (μg) A260/A280 A260/A230 1 5.7 1.84 1.53 2 5.2 1.86 1.7 3 5.6 1.87 1.76 4 5.4 1.88 1.76 5 5.5 1.87 1.69 6 5.7 1.82 1.08 Figure4. Comparison of extracted genomic DNA from E. coli purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) M 1 2 3 4 5 6 7 M: Bioneer 1kb ladder 1-2: (Mini) Extracted genomic DNA from E.coli (1x10 9 ) purified with Bioneer MagListo 5M Genomic DNA Extraction Kit 3-4: (Mini) Extracted genomic DNA from E.coli (1x10 9 ) purified with competitor Q kit 5-6: (Midi) Extracted genomic DNA from E.coli (5x10 9 ) 7: (Maxi) Extracted genomic DNA from E.coli (1x10 10 ) Sample Total Yield (ug) A260/A280 A260/A230 1 14.4 1.84 1.49 2 15 1.82 1.39 3 15.6 2.05 1.45 4 15.3 2.04 1.55 5 94 1.76 1.51 6 73.8 1.84 1.37 7 141.6 1.78 1.51 33
Figure5. Comparison of extracted genomic DNA from B. subtilis purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) M 1 2 3 4 5 6 M: Bioneer 1kb ladder 1-2: (Mini) Extracted genomic DNA from B. subtilis (1x10 9 ) purified with Bioneer MagListo 5M Genomic DNA Extraction Kit 3-4: (Mini) Extracted genomic DNA from B. subtilis (1x10 9 ) purified with competitor Q kit 5: (Midi) Extracted genomic DNA from B. subtilis (5x10 9 ) 6: (Maxi) Extracted genomic DNA from B. subtilis (1x10 10 ) Sample Total Yield (μg) A260/A280 A260/A230 1 15.5 1.96 2.17 2 15 1.91 2.11 3 9.7 1.96 1.15 4 11.7 1.99 1.31 5 45.7 1.94 2 6 56.6 1.82 2.07 Figure6. Comparison of Real Time qpcr data of extracted genomic DNA from HeLa cell (1x10 1 ~1x10 5 ) purified with MagListo 5M Genomic DNA Extraction Kit and competitor s kit (Single Column Type) 34
XVIII. 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 35
XIX. Explanation 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 36