Version No.: 2.0 ( )

Transcription

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2 Kit for the extraction of fragment DNA from agarose gel 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 X. Principle 4 XI. Magnetic Nano Bead Information 5 XII. Guidelines for MagListo Magnetic Separation Rack 5 XIll. Materials and Equipment Needed But Not Provided 6 Types of the Magnetic Separation Rack 6 XlV. Procedure 7 XV. Protocol 8 Fragment DNA Extraction from Gel 8 Summary of Reagents Volume in Each Step 11 XVI. Appendix 12 Troubleshooting guide 12 Experimental data 14 XVII. Ordering Information 15 XVIII. Explanation of Symbols 16

4 I. Overview Description MagListo 5M Gel Extraction Kit utilizes Magnetic Nano Beads to extract up to 10 μg of fragment DNA from agarose gel with 1X TAE or TBE buffer within 15 min, 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. The maximum amount of gel slice per each sample is about 400mg. The size range for effective extraction about 100 bp to 10 Kb and the average recovery yield exceeds 90% to 100%. Features and Benefits - Magnetic Nano Beads enable rapid extraction of fragment DNA - No requirement of expensive instruments Applications Sub-cloning, Sequencing, Labeling, DNA concentration and other molecular biological applications II. Kit Components MagListo 5M Gel Extraction Kit K-3607 (100 reaction) Buffer 1 (Gel Solubilization) 60 ml x 2 ea Buffer 2 (1 st Washing) 80 ml x 1 ea Buffer 3 (2 nd Washing) 80 ml x 1 ea Buffer 4 (3 rd Washing) 80 ml x 1 ea Buffer 5 (Elution) 15 ml x 1 ea Magnetic Nano Bead - DNA 1.8 ml x 6 ea 1

5 III. Storage MagListo 5M Gel 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 Gel Extraction Kit is intended for research use only. This kit is not intended for human or veterinary diagnostics. V. Safety Warnings and Precautions The Buffer l (Gel solubilization) should be stored in a dark place and redissolved before use. The Buffer 1 in the MagListo 5M Gel Extraction Kit is poisonous and hazardous. Please handle with care while working with Buffer 1 by wearing gloves and goggle eye protection. 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 2

6 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 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.co.kr 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 Gel Extraction Kit is carefully tested by the quality control team. 3

7 IX. Kit Specifications Gel slice ~ 400 mg Size range Binding capacity 100 bp - 10 kb ~ 10 g Typical recovery 90%-100% Expected purity A 260 / 280 > 1.8 Purification time < 15 min X. Principle MagListo 5M Gel Extraction Kit is designed for the extraction of highly purified fragment DNA from agarose gel with 1X TAE or TBE buffer. The overall principle is based on adsorption of DNA onto the Magnetic Nano Bead by chaotropic and other salt components which also enhance melting of agarose gel. For example, chaotropic agents in Buffer l (Gel Solubilization) contains such as guanidine thiocyanate, as which removes water molecules around DNA and silica coated magnetic beads surface resulting in fragment 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 4 (Elution), an aqueous solution with optimal ph. Gel Solubilization Binding Washing Elution 4

8 XI. 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 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 Silica Magnetic Nano Beads Average size Ligand 400nm -OH Working Temp Storage Store at room temperature upon receipt XII. Guidelines for MagListo Magnetic Separation Rack Description MagListo Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic NanoBeads. 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. 5

9 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. XIll. Materials and Equipment Needed But Not Provided 1. UV light-box 2. Scalpel ml tube or 2 ml tube 4. Vortex mixer 5. Absolute ethanol 6. Thermal block 7. MagListo Magnetic Separation Rack Types of the Magnetic Separation Rack Tube 1 ml tube with 8-cap strip MagListo Magnetic Separation Rack MagListo -8Ch Magnetic Separation Rack (Cat. no. TM-1000) 1.5 ml or 2 ml microcentrifuge tube MagListo -2 Magnetic Separation Rack (Cat. no. TM-1010) (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. 6

10 XIII. Procedure-Gel Extraction 7

11 XV. Protocol A. Fragment DNA Extraction from Gel 1. (Gel Cutting) Visualize the band, in an ethidium bromide stained gel, in a dark room with an UV light. Cut around the band of interest DNA using a scalpel blade. Switch off the UV light-box, carefully remove the slice from the gel and weigh the gel slice in a 1.5 ml or 2 ml tube (not provided). (Note) Set the UV light-box to long wave length UV, if possible, and minimize the exposure time to DNA. 2. (Gel Solubilization: 2-3) The maximum amount of gel slice per each sample is ~ 400 mg. Add 3 volumes of Buffer 1 (Gel Solubilization) to 1 volume of the gel slice. (If the weight of gel slice is 200 mg, add 600 μl of Buffer 1.) 3. Incubate at 60 for 10 min. Mix by inverting the tube every 2-3 min during the incubation. If the gel slice is dissolved incompletely, increase the incubation time. After dissolving the gel slice, check the color of the mixture whether it is yellow or not. (Note) The color of the mixture indicates ph of the mixture related with DNA binding. ph 7.5 (yellow color), the fragment DNA can effectively bind to the magnetic nano beads. 4. (DNA Binding with Magnetic Nano Bead: 4-6) Add 100 μl 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. 5. Place the tubes on the MagListo -2 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

12 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. 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. When discarding the supernatant, invert the rack completely so that the solution odes not to spill on the rack. 7. (1 st Washing: 7-9) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 700 μl of Buffer 2 (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. 8. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 9. Without removing the tubes from the MagListo Magnetic Separation Rack, discard the supernatant and completely remove the remaining supernatant on a paper towel by blotting action. 9

13 10. (2 nd Washing) Repeat the steps 7-9 by adding 700 μl of Buffer 3 (2 nd Washing) instead Buffer 2 for additional washing. 11. (3 rd Washing) Without removing the tubes from the MagListo Magnetic Separation Rack, add 700 μl 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 DNA yield than expected 12. Discard the supernatant and completely remove the remaining supernatant by blotting action. - Add Buffer 4 and discard the supernatant 13. (Elution: 13-17) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 30 ~ 50 μl of Buffer 5 (Elution) or distilled water to the each tube and resuspend the bead pellet completely by pipetting or using a vortex mixer for 15 sec. 14. Incubate the tube at 60 for 1 min. 15. Vortex the tube for 15 sec. 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 tube from the MagListo Magnetic Separation Rack, transfer supernatant containing DNA carefully to a new sterile microcentrifuge tube. 18. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads. 10

14 Summary of Reagents Volume in Each Step Step Buffer Volume Page Gel Solubilization Buffer 1 (Gel Solubilization) ~ 1200 μl P. 8 Bead Binding Magnetic Nano Bead - DNA 100 μl P. 8 1 st Washing Buffer 2 (1 st Washing) 700 μl P. 9 2 nd Washing Buffer 3 (2 nd Washing) 700 μl P rd Washing Buffer 4 (3 nd Washing) 700 μl P. 10 Elution Buffer 5 (Elution) μl P

15 XIV. Appendix 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. High ph conditions can reduce the overall yield due to incorrect binding conditions. This can be examined by using Buffer l (Gel Solubilization), which has a ph indicator. If the color is yellow but it turns to red or orange, then the ph is out of range. In this case, several drops of sodium acetate buffer are recommended to adjust the ph of the solution appropriately. Low recovery of DNA fragment Incomplete dissolving DNA of the gel slices gives lower yield. DNA can be remained in any undissolved agarose gel. Provide enough time for gel slice to be dissolved completely. Elution may have been incomplete. Please extend incubation time up to 3 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. 12

16 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 fragment DNA. Presence of a white precipitate in buffers A white precipitate may form in Buffer l (Gel Solubilization) due to prolonged storage at low temperatures. Incubate Buffer l at 60 to dissolve any precipitate in the buffer. 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. 13

17 Experimental data Figure 1. Comparison of Fragment DNA from TBE agarose gel purified with MagListo 5M Gel Extraction Kit and competitor s kit (Single Column Type) M M: Bioneer 1 Kb ladder 1-2: 500 bp DNA purified with Bioneer MagListo 5M Gel Extraction Kit 3-4: 500 bp DNA purified with competitor Q kit 5: Control 500 bp DNA 6-7: 3 kb DNA purified with Bioneer MagListo 5M Gel Extraction Kit 8-9: 3 kb DNA purified with competitor Q kit 10: Control 3 kb DNA 11-12: 5 kb DNA purified with Bioneer MagListo 5M Gel Extraction Kit 13-14: 5 kb DNA purified with competitor Q kit 15: Control 5 kb DNA 14

18 XVII. 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 15

19 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 16

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