Contents. XI. Materials and Equipment Needed But Not Provided 5. DNA Extraction from Small Amount of Tissue 10

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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. Product Specifications 4 Poly A (Carrier RNA) X. Principle 4 XI. Materials and Equipment Needed But Not Provided 5 XII. Protocols Before You Begin 6 DNA Extraction from Forensic Sample 6 DNA Extraction from Small Amount of Tissue 10 DNA Extraction from Urine 11 DNA Extraction from Small Volumes of Blood and Saliva 12 DNA Clean-Up 13 XIII. Troubleshooting guide 15 XIV. Ordering information 17 XV. Explanation symbols 18

4 I. Overview Description MagListo TM 5M Forensic Sample DNA Extraction Kit utilizes Magnetic Nano Beads to extract total DNA from a variety of forensic sample, such as whole blood, saliva, dried body fluid spot, fingerprint, nail clipping or hair using Magnetic Nano Beads and MagListo TM Magnetic Separation Rack. The use of MagListo TM Magnetic Separation Rack along with this kit greatly increases user s convenience by saving process time without use of centrifuge. Features and Benefits - Magnetic Nano Beads enable rapid extraction - No expensive instrument required. Our MagListo TM Magnetic Separation Rack is very cost conscious. Applications PCR, Real-Time PCR, SNP genotyping, STR (Short Tandem Repeat) analysis II. Kit Components MagListo TM 5M Forensic Sample DNA Extraction Kit Cat. no. 3614, 3615 K-3614 (8 reactions) K-3615 (100 reactions) Buffer 1 (Lysis) 4 ml x 1 ea 40 ml x 1 ea Buffer 2 (Binding) 3 ml x 1 ea 30 ml x 1 ea Buffer 3 (1 st Washing) 4 ml x 1 ea 40 ml x 1 ea Buffer 4 (2 nd Washing) 1.6 ml x 1 ea 16 ml x 1 ea Buffer 5 (Elution) 1 ml x 1 ea 15 ml x 1 ea Magnetic Nano Bead - DNA 1 ml x 1 ea 1.8 ml x 6 ea Proteinase K 5 mg x 1 ea 25 mg x 2 ea Poly A (Carrier RNA) - 1 mg x 1 ea 1

5 III. Storage MagListo TM 5M Forensic Sample DNA Extraction Kit should be stored dry at room temperature. It can be stored for up to 1 year, if it remains sealed. MagListo TM 5M Forensic Sample DNA Extraction Kit provides optimized Buffer 2 (Binding) which is poisonous and hazardous. Please, wear gloves and goggle eye protection when working with Buffer 2 (Binding). IV. Intended Use MagListo TM 5M Forensic Sample 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 Material Safety Data Sheet (MSDS) of 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 and tips should be processed and disposed of according to applicable and appropriate regulations of the municipality/government in which this product is being used. Users must be trained with basic experimental techniques for correct execution of the experiments described in the 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 unlicensed use of a patented application. 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 any issues are discovered relating to 2

6 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 in written form within 30 days of occurrence. VII. Technical Assistance At Bioneer, we pride ourselves on being responsive to customers needs. If you have any questions or would like to find out more information about MagListo TM products, please contact us. We look forward to hearing from you! Technical Support For all technical questions and troubleshooting on Bioneer products and applications. Tel: sales@bioneer.com In North America Tel: support@bioneer.us.com VIII. Quality Management Every aspect of our quality management system from product development, production to quality assurance and supplier qualification meets the world-class standards. Each lot of MagListo TM 5M Forensic Sample DNA Extraction Kit is carefully tested by our quality control team. 3

7 IX. Product Specifications Feature Sample type Technology Hands-on time Specification Various kind of forensic sample Magnetic Nano Bead < 10 min Expected purity A 260 / 280 > 1.8 Poly A (Carrier RNA) MagListo TM 5M Forensic Sample DNA Extraction Kit is able to extract total DNA from very small amounts of sample. If the sample contains low copy of cells (< 1x10 4 ) or has a small amount of DNA (<100 ng), we recommend adding 2 μg of poly A (KB ) to the sample after Buffer 2 (Binding). Carrier RNA, such as poly A, enhances binding of DNA to Magnetic Nano Beads. To prepare poly A solution please add 1 ml of nuclease free water to the tube containing lyophilized poly A (KB ) to obtain a solution of 1 μg/μl. Dissolve poly A completely, divide it into conveniently sized aliquots and store at 20 C. Freeze-thaw repetition of the aliquots more than 3 times will result in degradation of RNA. Poly A can be removed later by RNase digestion. If the sample contains large amount of DNA, addition of poly A is not necessary. X. Principle MagListo TM 5M Forensic Sample DNA Extraction Kit is designed for extraction of total DNA from a variety of sources including high molecular weight up to 40 Kb (Note: this is common for most DNA based application). Overall principle is based on adsorption of DNA onto Magnetic Nano Beads by chaotrophic salt. For instance, chaotropic agent in Buffer 2 (Binding) contains guanidine hydrochloride. This removes water molecules around DNA and silica coated Magnetic Nano Beads surface, resulting in total DNA being captured by Magnetic Nano Beads. Magnetic Nano Beads and nucleic acid complexes are then pulled and fixed on the tube wall using a magnetic force, followed by washing with ethanol to remove debris and excessive salts. Captured nucleic acids are then eluted by Buffer 5 4

8 (Elution), which contains an aqueous solution with optimal ph. Sample Lysis Binding Washing Elution XI. Materials and Equipment Needed But Not Provided 1. 1 ml tube with 8-cap strip, 1.5 ml or 2 ml tubes based on sample size and MagListo TM Magnetic Separation Rack 2. Vortex mixer 3. Absolute ethanol 4. Thermal block or dry oven 5. Phosphate-buffered saline (PBS) 6. Blow dryer or heat gun 7. MagListo TM Magnetic Separation Rack with appropriate size Magnetic Separation Rack Choice Tube 1 ml tube with 8-cap strip 1.5 ml or 2 ml microcentrifuge tube MagListo TM Magnetic Separation Rack MagListo TM -8Ch Magnetic Separation Rack (Cat. #. TM-1000) MagListo TM -2 Magnetic Separation Rack (Cat. #. TM-1010) Please refer to the ordering information table on the latter part of the manual, which contains the appropriate catalog number for specific tube size. 5

9 XII. Protocols Before you begin 1. Completely dissolve Proteinase K in 250 ul (KB ) or 1,250 ul (KB-0111) of nuclease-free water. Dissolved Proteinase K should be stored at Completely dissolve Poly A (KB ) in 1,000 ul of nuclease-free water. Divide it into conveniently sized aliquots and store at 20 C. 3. Buffer 2 (Binding) contains chaotropic salt. You should take the appropriate laboratory safety precautions and wear gloves when handling. 4. Add correct amount of absolute ethanol to Buffer 3 (1 st Washing) and 4 (2 nd Washing). a. DNA Extraction from Forensic Sample Handling Samples Dried body fluid spot or fingerprint (FTA card, paper, cloth etc.) Punch out the sample up to 7 mm diameter using single-hole paper puncher or cut out up to 2 cm 2. Cut the sample into smaller pieces to increase lysis efficiency. Hair Cut the hair 1 cm length from the hair root. Without root, cut up to 5 strands of whole hair into small pieces. Bone & teeth Homogenize the bone up to 100mg into fine powder. Chewing gum Cut up to 30 mg of chewing gum into small pieces. Cigarette butts Cut out up to 2 cm 2 piece of outer paper from the end of the cigarette butt. Buccal swab Cut the swab from its stick by hand or scissors. Use single piece of swab for extraction. 1. Apply the forensic sample to a 1.5 ml or 2 ml tube. 6

10 2. (Lysis: 2-7) Add 300 μl of Buffer l (Lysis) and 10 μl of proteinase K solution (see Before you begin ) to the tube and completely resuspend the contents with vortex mixer or pipetting. 3. (Optional) Add 20 μl of 1M DTT (Dithiothreitol, Not provided) to the tube and completely resuspend the contents with vortex mixer. If the sample is hair, nail clipping or semen stains, this step is necessary to increase sensitivity. 4. Incubate at 60 for at least 1 hour. (Note) Incubation time for complete lysis varies depending on the type of sample used and age of starting material. If the sample is nail clipping, bone or considerably old, extend incubation time up to overnight. 5. Add 300 μl of Buffer 2 (Binding) to the tube and completely resuspend the contents by vortex mixer or pipetting. 6. (Optional) Add 2 μl of dissolved poly A (see Before you begin or page 4 for details) to the tube. 7. Incubate at 60 for 20 min. 8. Centrifuge at 13,000 rpm for 1 min to obtain clear lysate. 9. Take the clear supernatant only and transfer into new 1.5 ml or 2 ml tube. 10. (DNA Precipitation) Add 600 μl of absolute ethanol and mix well using a vortex mixer or pipetting. 11. (DNA binding with Magnetic Nano Bead: 11-13) Add 100 μl of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until Nano Beads are fully resuspended. (Note) Magnetic Nano Bead solution should be shaken well before each use. 12. Place the tube in MagListo TM -2 Magnetic Separation Rack with magnet plate attached 7

11 and invert the Rack with tube 3-4 times gently until Nano Beads bind tightly to magnet. - Attachment Combine magnet plate and the stand. 13. Without removing the tube from MagListo TM rack, carefully pour supernatant out and completely remove the remaining supernatant using a paper towel. In process, magnetic crude pellet remains attached to the side of tube. - Discard solution Discard solution by inverting MagListo rack. Silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discarding solution, invert rack completely so the solution does not smear on the rack. 14. (1 st washing: 14-16) Detach magnet plate from MagListo TM stand. Add 700 μl of Buffer 3 (1 st Washing) to the tube. Close the cap and mix with vortex mixer until Nano Beads are fully resuspended. - Detachment Push magnet plate upward gently. 8

12 15. Attach the magnet plate to MagListo TM stand and invert the Rack with tube 3-4 times gently until Nano Beads bind tightly to magnet. 16. Without removing the tube from MagListo TM rack, pour supernatant out and remove remaining supernatant using a paper towel by blotting. 17. (2 nd washing) Repeat the above step by adding 700 μl of Buffer 4 (2 nd Washing) for additional washing. 18. (3 rd washing) Repeat the above step by adding 700 μl of absolute ethanol for additional washing. 19. (Drying) Completely dry Nano Beads with the tube open and use a heat gun or a blow dryer for 1 min 3 cm away from the top of the tube. (Note) If you do not have using a heat gun or a blow dryer, place the rack lying down in a dry oven at 60 for 10 min. 20. (Elution: 20-24) Add μl of Buffer 5 (Elution) or distilled water to the tube with magnet plate detached and resuspend the contents completely by pipetting or vortex mixer for 15 sec. 21. Incubate the tube at 60 for 1 min. 22. Attach magnet plate to MagListo TM stand and invert the Rack with tube 3-4 times gently until Nano Beads bind tightly to the magnet. 23. Without removing the tube from MagListo TM rack, carefully transfer supernatant containing DNA to a sterile microcentrifuge tube. 24. Discard used Magnetic Nano Beads. Do not reuse Nano Beads. 9

13 b. DNA Extraction from Small Amount of Tissue 1. (Homogenization) Disrupt (or homogenize) the sample (~ 10 mg) with homogenization tool. Place them into a 1.5 ml or 2 ml tube immediately. (Note) Grind the tissue completely into fine powder with mortar and pestle or other homogenization tool under liquid nitrogen. Final yield of DNA depends on the amount and the type of the used tissue. 2. (Lysis: 2-6) Add 90 μl of Buffer l (Lysis). 3. Add 10 μl of proteinase K solution (see Before you begin ) to the tube and mix thoroughly using a vortex mixer. 4. (Optional) If RNA-free DNA is required, add up to 200 ug of RNase A (KB-0101, not provided) and incubate for 2 min at room temperature. 5. Incubate at 60 until the tissue is completely lysed. 6. Add 100 μl of Buffer 2 (Binding) to the tube and mix immediately and thoroughly using a vortex mixer.. 7. (DNA Precipitation) Add 200 μl of absolute ethanol and mix well using a vortex mixer or pipetting. 8. Go to step 11 of DNA Extraction from Forensic Sample in page 7 and continue extraction process. 10

14 c. DNA Extraction from Urine 1. (Cell collection: 1-3) Centrifuge the urine and discard supernatant. a. (~ 2ml) Centrifuge at 8,000 rpm (6,000 xg) for 2 min. b. (~ 15ml) Centrifuge at 3,500 rpm (2,000 xg) for 10 min. 2. Add 500 ul of PBS and completely resuspend with vortex mixer or pipetting. Transfer the sample to a 1.5 ml or 2 ml tube. 3. Centrifuge at 8000 rpm for 2 min and discard supernatant. 4. (Lysis: 4-8) Add 300 μl of Buffer l (Lysis) and 10 μl of proteinase K solution (see Before you begin ) to the tube and completely resuspend the contents with vortex mixer or pipetting. 5. (Optional) Add 10 μl of 1M DTT to the tube and completely resuspend the contents with vortex mixer. If the urine is expected to contain sperm cells, this step will improve yield. 6. Incubate at 60 for 1 hour. 7. Add 300 μl of Buffer 2 (Binding) to the tube and completely resuspend by vortex mixer. 8. Incubate at 60 for 10 min. 9. (DNA Precipitation) Add 600 μl of absolute ethanol and mix well using a vortex mixer or pipetting. 10. Go to step 11 of DNA Extraction from Forensic Sample in page 7 and continue extraction process. 11

15 d. DNA Extraction from Small Volumes of Blood and Saliva 1. Add 10 μl of proteinase K solution (see Before you begin ) to 1ml, 1.5 ml or 2 ml tube. 2. Apply 1~100 μl of blood or saliva to the tube containing proteinase K. (Note) If the sample volume is less than 100 ul, make the total volume 100 μl by adding 1X PBS to achieve maximum lysis efficiency and yield. 3. (Lysis: 3-4) Add 100 μl of Buffer 2 (Binding) to each sample and mix immediately and thoroughly using a vortex mixer. You must completely resuspend the sample to achieve maximum lysis efficiency. 4. Incubate at 60 for 10 min. 5. (DNA Precipitation) Add 200 μl of absolute ethanol and mix well using a vortex mixer or pipetting. 6. Go to step 11 of DNA Extraction from Forensic Sample in page 7 and continue extraction process. 12

16 e. DNA Clean-Up 1. Transfer the eluate or enzyme reaction product to 1.5 ml or 2 ml tube. 2. (Optional) If RNA-free DNA is required, add up to 200ug of RNase A (KB-0101, Not provided) and incubate for 2 min at room temperature. 3. (Binding) Add 1 volume of Buffer 2 (Binding) to the eluate and mix completely with vortex mixer. 4. (DNA precipitation) Add 3 volumes of absolute ethanol to the eluate and mix well with vortex mixer. 5. (DNA binding with Magnetic Nano Bead: 5-7) Add 100 μl of Magnetic Nano Bead solution to the tube and mix thoroughly using a vortex mixer until Nano Beads are fully resuspended. (Note) Magnetic Nano Bead Solution contains magnetic nano beads. Please shake well before use. 6. Place the tube in MagListo -2 Magnetic Separation Rack with the magnet plate attached and invert the Rack with tube 3~4 times gently until Nano Beads bind tightly to magnet. - Attachment Combine magnet plate and the stand. 7. Without removing the tube from MagListo rack, carefully pour supernatant out and completely remove remaining supernatant using paper towel by blotting. 13

17 - Discard solution Discard solution by inverting the MagListo rack. Silicone immobilizer inside the stand holds the tubes from falling in an upside down position. When discarding solution, invert rack completely so the solution does not smear on the rack. 8. (1 st washing: 8-10) Detach the magnet plate from MagListo stand. Add 700 μl of Buffer 4 (2 nd Washing) to the tube. Close the cap and mix with vortex mixer until Nano Beads are fully resuspended. - Detachment Push magnet plate upward gently. 9. Attach magnet plate to MagListo stand and invert the tube 3~4 times gently until beads bind tightly to magnet. 10. Without removing the tube from MagListo rack, pour supernatant out and remove remaining supernatant using paper towel by blotting. 11. Go to step 18 of DNA Extraction from Forensic Sample in page 9 and continue cleanup process. 14

18 XIII. Troubleshooting Guide Comments and suggestions Buffers or other reagents may have been exposed to external changes or conditions that reduce its effectiveness. Please make sure that reagents were stored at room temperature at all times upon arrival and all reagent bottles were closed tightly, in order to preserve ph and stability and to avoid contamination. Please check and add ethanol to the Buffer 3 (1 st Washing) and 4 (2 nd Washing). After adding ethanol, mix Washing Buffer well and always mark the Washing Buffer bottles. If not, Washing Buffer remain concentrated and may wash away the adsorbed DNA. Low yield of DNA The lysis may have been incomplete. Please extend the incubation time. The amount of time for complete lysis varies depending on the type of sample used and age of starting material. A shaking water bath should be used for efficient lysis. Elution may have been incomplete. Please extend incubation time up to 3 minutes at elution step to improve yield. In addition, make sure that Magnetic Nano Beads are resuspended completely in the eluate during incubation. Pellet of Magnetic Nano Bead could be lost while discarding solution. Check that all of the Nano Beads bind tightly to magnet when you discard solution. 15

19 Beads may have been dried insufficiently. You must completely dry Nano Beads in a drying step. Remained ethanol can decrease purity of DNA. Take enough time to completely dry Nano Beads. Low A 260/280 ratio Incomplete suspension of beads during the washing step causes salts to remain in purified DNA. Make sure Nano Beads are suspended thoroughly during the washing process. MagListo 5M Forensic Sample DNA Extraction Kit copurify DNA and RNA when both are present in the sample. If RNA-free DNA is Copurification of RNA required, 200ug of RNase A (KB-0101, not provided) should be added to each sample before addition of Buffer 2 (Binding). If you want to remove RNA in the eluate, refer to DNA Clean-Up protocol in page 13 for details. There is a white precipitate in some buffer A white precipitate may form in Buffer l (Lysis) or Buffer 2 (Binding) after prolonged storage at low temperature. Incubation at 60 should dissolve any precipitate in Buffer l or Buffer 2. 16

20 XIV. Ordering Information Cat no. Product Description Size K-3601SM MagListo TM 5M Plamid Extraction Kit, 8 reactions (mini) 1 kit 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-3602 MagListo TM 5M Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3603 MagListo TM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3604 MagListo TM 5M Plant Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit K-3605 MagListo TM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit K-3606 MagListo TM 5M Gel Extraction Kit, 8 reactions (mini) 1 kit K-3607 MagListo TM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit K-3608 MagListo TM 5M PCR Purification Kit, 8 reactions (mini) 1 kit K-3609 MagListo TM 5M PCR Purification Kit, 100 reactions (mini) 1 kit K-3610 MagListo TM 5M Cell Total RNA Extraction Kit, 8 reactions (mini) 1 kit K-3611 MagListo TM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit K-3614 MagListo TM 5M Forensic Sample DNA Extraction Kit, 8 reactions (mini) 1 kit K-3615 MagListo TM 5M Forensic Sample DNA Extraction Kit, 100 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 TM-1030 MagListo TM -15 Magnetic Separation Rack MagListo TM -50 Magnetic Separation Rack 15 ml tube x 6 holes 50 ml tube x 3 holes TM-1040 MagListo TM -96 Magnetic Separation Rack 96-well plate 1ea TM-1100 MagListo TM Magnetic Separation Rack Bundle Set MagListo TM -2, -15, -50, and -96 (4 racks, 1 each) 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 17

21 XV. Explanation Symbols Catalog Number Contains sufficient for (n) tests USE BY Consult Instruction For Use Batch code Caution, consult accompanying documents Temperature Limitation Research Use Only Manufacturer Caution, Potential Biohazard DO NOT REUSE 18

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