Kit for the extraction of plasmid from bacterial cultures 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 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. Kit Specifications 4 X. Sample Preparation 4 XI. Principle 6 XII. Magnetic Nano Bead Information 6 XIII. Guidelines for MagListo Magnetic Separation Rack 7 XIV. Materials and Equipment Needed But Not Provided 8 Types of the Magnetic Separation Rack 8 XV. Procedure 9 XVI. Protocols 10 Before You Begin 10 Bacteria Culture and Collection 10 Plasmid Extraction Protocol for Mini/Midi/Maxi/8 channel 11 Summary of Reagents Volume in Each Step 14
XVII. Appendix 15 Troubleshooting guide 15 Experimental data 18 XVIII. Ordering Information 20 XIX. Explanation of Symbols 21
I. Overview Description MagListo 5M Plasmid Extraction Kit utilizes Magnetic Nano Beads to extract plasmid DNA rapidly from bacterial cultures 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, Sequencing, Transformation, Transfection, In-vitro Transcription/Translation II. Kit Components MagListo 5M Plasmid Extraction Kit *K-3601 **K-3600 Buffer 1 (Resuspension) 25 ml x 1 ea 110 ml x 1 ea Buffer 2 (Lysis) 25 ml x 1 ea 110 ml x 1 ea Buffer 3 (Neutralization) 25 ml x 1 ea 110 ml x 1 ea Buffer 4 (Binding) 75 ml x 1 ea 180 ml x 2 ea Buffer 5 (3 rd Washing) 120 ml x 1 ea 250 ml x 2 ea Buffer 6 (Elution) 15 ml x 1 ea 50 ml x 1 ea Magnetic Nano Bead 1.2 ml x 5 ea 30 ml x 1 ea RNase A powder, lyophilized 3 mg x 1 ea 12 mg x 1 ea *mini 100 rxn, midi 10 rxn, maxi 4 rxn **mini 500 rxn, midi 50 rxn, maxi 18 rxn 1
III. Storage MagListo 5M Plasmid Extraction Kit should be stored at room temperature. It can be stored for up to 2 years 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. After addition of RNase A powder, Buffer 1 (Resuspension) is stable for 6 months when stored at 2-8. RNase A powder can be stored for two years at room temperature. A white precipitate may form in Buffer 2 (Lysis) or Buffer 4 (Binding) after prolonged storage at low temperature. Incubating at 60 should dissolve any precipitate in Buffer 2 (Lysis) or Buffer 4 (Binding). IV. Intended Use MagListo 5M Plasmid Extraction Kit is intended for research use only. This kit is not intended for human or veterinary diagnostics. V. Safety Warnings and Precautions Please inquire BIONEER s Customer Service Center to obtain a copy of the Material Safety Data Sheet (MSDS) for this product. Before, during and after using this kit, all potentially hazardous materials (i.e. materials that may have come in contacted with genetically recombinant samples) including tubes, tips and other kit contents should be processed and discarded in accordance with applicable and appropriate regulations of the municipality/government in which this product is being used. A user must also be equipped with basic experimental techniques required for correct execution of the extraction experiments described in this User s Guide. Some inventions applied in this kit may infringe existing patents in certain countries. The purchase of this kit does not include or provide a license to perform such patented inventions. Users may be required to obtain a license depending on the patent law of the country where this product is being used. We do not condone nor recommend the unlicensed use of patented inventions. 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 (1) year from the date of purchase. If any issues are discovered relating to compromise in product quality, immediately contact BIONEER s Customer Service Center (sales@bioneer.com). BIONEER does not assume any liability for the 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. 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, production to quality assurance and supplier qualification meets the world-class standards. Each lot of MagListo 5M Plasmid Extraction Kit is carefully tested by the quality control team. 3
IX. Kit Specifications Scale Micro scale Mini scale Midi scale Maxi scale Starting culture volume 1-1.5 ml 1-5 ml 20-50 ml 100-200 ml Preparation time < 20 min < 5 min < 10 min < 15 min Elution volume 100 µl 100 µl 500 µl 1 ml Expected DNA yield Up to 10 µg Up to 20 µg Up to 200 µg Up to 500 µg Expected purity A 260 / 280 > 1.8 * The yield of low copy plasmid could be less than the amount shown in this table. X. Sample Preparation Starting material Several factors such as plasmid copy number, bacterial strain, antibiotics, inoculation and type of culture medium may influence the yield of plasmid DNA. It is recommended to extract plasmids from the bacterial cultures that have been inoculated with a single pure colony from the agar plate. Copy number of plasmid Plasmids used in molecular cloning usually contain pmb1 derived replicons (e.g. puc plasmid). Plasmids that contain pmb1 derived replicons have a relaxed control of replication and thus have very high copy numbers. In contrast, psc101 plasmid has a stringent control of replication and maintain a low copy number. Generally, high-copy number plasmids will result in higher yield than low-copy number plasmids. Table 1. Replicons carried by various plasmid vectors Plasmids Copy number Replicon Classification puc 500-700 pmb1* High copy pgem 300-500 pmb1* High copy pbluescript 300-500 ColE1* High copy ptz >1000 pmb1* High copy pet 15-20 pmb1* Low copy 4
payc 10-12 P15A Low copy pbr322 15-20 pmb1* Low copy psc101 ~5 psc101 Very low copy * The pmb1 origin of replication (ori) is closely related to that of ColE1 and belongs in the same incompatibility group of ColE1. The high-copy plasmids listed above contain pmb1 derived replicons. Host strains Some strains, especially enda+ strains such as JM101, JM110, HB101, TG1 and their derivatives may produce lower yield and quality of plasmid DNA than DH1, DH5α and XL1-Blue strains. In addition, strains derived from HB101 strains can produce large amounts of carbohydrates, which may inhibit activity of many restriction enzymes and polymerases. Antibiotics Antibiotics used in bacterial selection can influence growth of bacteria at all stages. As antibiotics are sensitive to storage condition and high temperatures, stock solutions of antibiotics should be stored in aliquots at -20. To test whether antibiotics are still effective, fresh cultures inoculated from freshly prepared bacterial culture plates should be used. Table 2. Recommended concentrations of commonly used antibiotics Antibiotics Stock solution Storage Working concentration Ampicillin 40mg/ml in H 2O -20 80μg/ml Tetracycline HCl 10mg/ml in 50% Ethanol -20 50μg/ml Kanamycin 10mg/ml in H 2O -20 50μg/ml Culture medium The Luria Bertani (LB) medium is one of the most commonly used media to grow bacterial cells. MagListo 5M Plasmid Extraction Kit is optimized for use with LB media. As the ratio of plasmid DNA to RNA is higher during the logarithmic phase, it is recommended to collect bacterial cells cultured for approximately 12 h to 16 h (when stationary growth phase starts). 5
XI. Principle MagListo 5M Plasmid Extraction Kit is designed for the extraction of highly purified plasmid from cultured bacterial cells as fast as 5 min. The overall principle is based on the modified alkaline lysis method (Birnboim et al, 1979) and Bioneer s novel Nano-Technology (Patent pending). Collected cells are re-suspended in Buffer 1 (Resuspension) that contains Nano Beads, followed by a lysis step with the addition of Buffer 2 (Lysis). When Buffer 3 (Neutralization) is added to the lysate, the chromosomal DNA and cell debris forms insoluble aggregates. While forming aggregates, Bioneer s Nano Beads effectively facilitate the process by binding to protein and cell debris aggregates and increasing the total weight of the complex. The aggregates are separated using a magnetic force from supernatant including plasmid, which is then transferred to a new tube for an ensuing process. Guanidine hydrochloride, as chaotropic agents in Buffer 4 (Binding), removes water molecules around DNA and silica coated magnetic bead surface resulting in plasmid then being captured by silica coated magnetic beads. The magnetic bead and nucleic acid complexes are pulled and fixed on the tube wall using a magnetic force, followed by washing with 80% ethanol to remove debris and excessive salts. The captured nucleic acids are then eluted by Buffer 6 (Elution), an aqueous solution with 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 with DNA and the Magnetic Nano Beads are then isolated using external magnetic field. 6
Features Fast binding guarantees higher throughput automation Large Surface Area enables more sensitive assay Globular structure increases specificity by decreasing non-specific binding Specification AccuNanoBead Silica Magnetic Nano Beads Matrix Silica-coated Fe 3 O 4 Average size Ligand 400nm -OH Working Temp. 0-100 Storage Store at room temperature upon receipt XIII. 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. When an extracted or purified nucleic acid is spilled on the product, immediately rinse it with running water and clean it with 70% ethanol. Acetone, Toluene, or organic solvent may damage the acrylic and plastic part of the product, which may lead to malfunction of the product. Rinse the product immediately when spillage of any above mentioned solvents occurs as the expected DNA yield may not be obtained if the product is damaged. Make sure that a corrosive liquid on the magnet plate part of the product. If spillage occurs, immediately rinse it off with running water as it may corrode the magnet during storage and may 7
degrade its performance. XIV. Materials and Equipment Needed But Not Provided 1. Centrifuge with rotor capable of 3,000 x g (midi & maxi scale) 2. 2 ml tube (mini scale), 15 ml tube (midi scale), 50 ml tube, (maxi scale), 1 ml tube (8-channel prep) 3. 96-well plate 4. Vortex mixer 5. 80% ethanol 6. MagListo Magnetic Separation Rack 7. (Optional) Table-top microcentrifuge, 16,000 x g (>13,000 rpm) (mini scale), High speed centrifuge (capable of 12,000 rpm, e.g., Beckman Avanti J Series, Sorvall RC-5B Plus, Hanil Supra series) (midi, maxi scale) 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-Plasmid Extraction 9
XVI. Protocols Before You Begin 1. Completely dissolve RNase A powder in Buffer 1 (Resuspension). After addition of RNase A powder, Buffer 1 (Resuspension) is stable for 6 months when stored at 2-8. 2. Buffer 4 (Binding) contains chaotropic salt. You should take the appropriate laboratory safety precautions and wear gloves when handling. 3. 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. 4. If there is any precipitate in Buffer 2 (Lysis), incubate on a heating block at 56 Bacterial Culture and Collection For mini prep / 8-channel prep 1. Pick up a single pure colony from freshly cultured LB (Luria-Bertani) agar plate or your media of choice containing antibiotics, and inoculate into 1 ml to 5 ml of fresh LB liquid media or your liquid media in a shaking incubator at 37 for 12 h to16 h. (Note) The overgrowth of E. coli cells may cause cell death, which may lead to lower yield of plasmid DNA due to inefficient lysis. For high copy number plasmid: 1-5 ml of E. coli cells. For low copy number plasmid: 2-10 ml of E. coli cells. 2. After incubation, collect the E. coli cells by centrifuging at 6000 x g for 15 min at 4. Remove the supernatant (liquid media) completely using a paper towel by blotting action. 3. Continue with Plasmid Extraction Protocol in page 10 for extraction of plasmids. For midi prep / maxi prep 1. Pick up a single pure colony from freshly cultured LB (Luria-Bertani) agar plate or your media of choice containing antibiotics, and inoculate into 1 ml to 5 ml of fresh LB liquid media in a shaking incubator at 37 for 12 h to 16 h. 2. After incubation, re-inoculate 50 μl of cultured liquid media into 20-50 ml (midi) / 100-200 ml (maxi) of fresh LB liquid media or your liquid media of choice containing antibiotics. Incubate the inoculated liquid media in a shaking incubator at 37 for 12 h to 16 h. 3. After incubation, collect the E. coli cells by centrifuging at 6 000 x g for 15 min at 4. Remove the supernatant (liquid media) completely using a paper towel by blotting action. 4. Continue with Plasmid Extraction Protocol in page 10 for extraction of plasmids. 10
Plasmid Extraction Protocol for Mini / Midi / Maxi / 8-channel 1. (Resuspension of E. coli pellet) Add 200 μl (mini) / 2 ml (midi) / 6 ml (maxi)/ 100 μl (8-channel) of Buffer 1 (Resuspension) to the harvested cell pellet and completely resuspend by vigorous vortexing or several pipetting. (Note) You must completely resuspend the cell pellet to achieve maximum lysis efficiency. Given that buffer 1 (Resuspension) contains Magnetic Nano Beads, please shake well the bottle before use. If you have harvested cell pellet in a 15 ml (mini) / 50 ml (midi) / 100-250 ml (maxi) / 1.5 ml (8-channel) tube, please transfer the resuspended cells to a 2 ml or 1.5 ml (mini) / 15 ml (midi) / 50 ml (maxi) / 1 ml (8-channel) tube. *Caution! Please check the magnetic bead completed suspended in solution. Incomplete suspension can lead to partial removal of aggregation. 2. (Lysis of cell) Add 200 μl (mini) / 2 ml (midi) / 6 ml (maxi) / 100 μl (8-channel) of Buffer 2 (Lysis) to the each tube and mix by inverting the tube 3-4 times gently. (Note) Do not vortex but just invert gently. Vortexing can cause shearing of genomic DNA. 3. (Neutralization) Add 200 μl (mini) / 2 ml (midi) / 6 ml (maxi) / 100 μl (8-channel) of Buffer 3 (Neutralization) to the each tube and immediately mix by inverting the tube 3-4 times gently. (Note) Genomic DNA and cell debris will form an insoluble complex with Nano Beads in this step. Again, be cautious to handle the lysate gently not to shear genomic DNA. 4. (Debris removal: 4-6) Place the tube on the MagListo -2 (mini) / -15 (midi) / -50 (maxi) /-8Ch (8- channel) rack Magnetic Separation Rack with the magnet plate attached and invert the rack gently 3-4 times until the beads bind tightly to magnet. (Optional) For better yield, centrifuge the tube at 13,000 rpm for 1 min (mini) / 5 min (midi) / 10 min (maxi). Transfer cleared lysate to a new 2 ml or 1.5 ml (mini) / 15 ml (midi) / 50 ml (maxi) tube and go step 7. - Attachment of the magnet plate Combine the magnet plate to the stand. 11
5. Without removing the tube from MagListo Magnetic Separation Rack, discard the supernatant carefully to a new 2 ml (or 1.5 ml) (mini) / 15 ml (midi) / 50 ml (maxi) / 1 ml (8-channel) tube. 6. Repeat the above step 4 and 5 for clearer supernatant. We strongly recommend the repetition for better results. 7. (Plasmid binding with Magnetic Nano Bead: 7-9) Add 600 μl (mini) / 6 ml (midi) / 18 ml (maxi) / 300 μl (8-channel) of Buffer 4 (Biding) and then add 50 μl (mini)/ 300 μl (midi)/ 1.5 ml (maxi)/ 25 μl (8- channel) of evenly mixed Magnetic Nano Bead solution to the each tube. Close the cap and mix by inverting the each tube 3-4 times with the magnet plate detached. (Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well before use. 8. Place the tube on the MagListo Magnetic Separation Rack with the magnet plate attached and invert the rack gently 3-4 times until the beads bind tightly to magnet. 9. Without removing the each tube from MagListo Magnetic Separation Rack, discard the supernatant carefully completely remove the remaining supernatant on a paper towel by using 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. 10. (Washing: 10-12) Detach the magnet plate from MagListo Magnetic Separation Rack. Add 1 ml (mini) / 10 ml (midi) / 30 ml (maxi) / 500 μl (8-channel) of 80% ethanol to the tube and close the cap. Mix by inverting until the beads are fully resuspended. - Detachment of the magnet plate 12
Detach the magnet plate gently by pulling it upwards. 11. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the magnet. 12. Without removing the tube from MagListo Magnetic Separation Rack, discard the supernatant and completely remove the remaining supernatant on a paper towel by blotting action. 13. Repeat the above step 10-12 by adding 1 ml (mini) / 10 ml (midi) / 30 ml (maxi) / 500 μl (8-channel) of 80% ethanol for additional washing. 14. (3 rd Washing) Without removing the tubes from the MagListo Magnetic Separation Rack, add 700 μl (mini, 8-channel) / 10 ml (midi) / 30 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 results in lower DNA yield. 15. Discard the supernatant and completely remove the remaining supernatant by blotting action. - Add Buffer 5 and discard the supernatant 16. (Elution: 16-20) Detach the magnet plate from the MagListo Magnetic Separation Rack. Add 100 μl (mini, 8-channel) / 500 μl (midi) / 1 ml (maxi) of Buffer 6 (Elution) or distilled water to the each tube with the magnet plate detached and resuspend by tapping the tube wall. 13
17. Incubate the tubes at 60 for 1 min. 18. 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. 19. Without removing the tube from the MagListo Magnetic Separation Rack, transfer supernatant containing DNA carefully to a new sterile microcentrifuge tube. 20. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads. Summary of Reagents Volume in Each Step Step Buffer 8-channel Mini Midi Maxi Page Sample collection Culture volume 1-1.5 ml 1-10 ml 20-50 ml 100-200 ml P. 10 Resuspension Buffer 1 100 μl 200 μl 2 ml 6 ml P. 11 Lysis Buffer 2 100 μl 200 μl 2 ml 6 ml P. 11 Neutralization Buffer 3 100 μl 200 μl 2 ml 6 ml P. 11 Plasmid Binding Buffer 4 300 μl 600 μl 6 ml 18 ml P. 12 Bead Binding Magnetic Nano Bead 25 μl 50 μl 300 μl 1.5 ml P. 12 1 st, 2 nd Washing 80% Ethanol 500 μl 700 μl 10 ml 30 ml P. 12 3 rd Washing Buffer 5 700 μl 700 μl 10 ml 30 ml P. 13 Elution Buffer 6 100 μl 100 μl 500 μl 1 ml P. 13 14
XVII. Appendix Troubleshooting guide The 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 Performance of buffers or other reagents may have been lowered. 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. Cell debris is not fully removed. Include debris removal step, especially for mini (8-Channel?) scale. Refer to step 4 (optional) in page 8 for detail. The cells may not have been completely resuspended with Buffer 1 (Resuspension). Make sure that cells are completely resuspended. Excess amount of starting sample was used to extract DNA. Add Low or no yield appropriate amount of starting sample (see Kit Specification in page 4) for efficient extraction of genomic DNA. Elution may have been incomplete. 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. 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. 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. 15
Beads may have been washed insufficiently. Wash the beads properly in the 3 rd washing step. Remaining ethanol can decrease the purity of DNA. Take enough time to properly wash the beads. Low A 260/280 ratio Incomplete suspension of beads during the washing step causes salts to remain in the purified DNA. Make sure that the beads are suspended thoroughly during the washing process. During neutralization step, it is important that you do not vortex or shake Contamination of chromosomal DNA sample tubes vigorously. Also, total lysis reaction should not be longer than 5 min. These conditions can shear chromosomal DNA. Make sure that the lysate was handled gently. Aggregation of Magnetic Nano Bead Excess amount of starting sample is used to extract DNA. Add appropriate amount of starting material (see Kit Specification in page 4) for efficient extraction of genomic DNA. Presence of white precipitate in some buffer A white precipitate may form in Buffer 4 (Binding) due to prolonged storage at low temperatures. Incubate Buffer 4 (Binding) at 60 to dissolve any precipitate in the buffer. Too many background bands in sequencing analysis There may be high endonuclease activity in your strain of host E. coli, such as HB101, JM series etc. EndA- strain instead of EndA+ strain instead. RNase A activity becomes lowered, especially in the case that it has been Sample containing RNA over 6 months since RNase A powder was added to Buffer 1 (Resuspension). Add more RNase A powder, up to 100 ng/μl, or use fresh RNase A. 16
The DNA from old or inappropriately stored sample may often be degraded. As the DNA yield is highly dependent on storage conditions of Degraded DNA samples, please use fresh samples for optimal results. In case of using stored tissue sample, it is recommended to use sample stored at -70. Frequent freezing and thawing may result in lower DNA yield. 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. 17
Experimental data Figure 1. Electrophoresis data of several sizes of plasmid purified with MagListo 5M Plasmid Extraction Kit M1 1 2 3 M2 M1: Bioneer 100bp ladder 1: 3.5 kb plasmid 2: 5.4 kb plasmid 3: 8 kb plasmid M2: Bioneer 1kb ladder Figure 2. Electrophoresis data of restriction endonuclease-treated plasmid (500 ng/lane) after extracted with MagListo 5M Plasmid Extraction Kit in three different scales: mini, midi and maxi Mini Midi Maxi M1 1 2 3 4 5 6 7 8 9 10 M2 plasmid insert M1: Bioneer 100 bp ladder 1, 3: Plasmid (mini prep) 2, 4: Plasmid (mini prep) digested by EcoRI 5, 7: Plasmid (midi prep) 6, 8: Plasmid (midi prep) digested by EcoRI 9: Plasmid (maxi prep) 10: Plasmid (miaxi prep) digested by EcoRI M2: Bioneer 1 kb ladder 18
Figure 3. High quality sequencing data (ABI3730) of pgem-b1 purified with MagListo 5M Plasmid Extraction Kit 19
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 20
XIX. Explanation of Symbols Catalog Number Batch code Manufacturer Contains sufficient for (n) tests Caution, consult accompanying documents Caution, Potential Biohazard USE BY Temperature Limitation DO NOT REUSE Consult Instruction For Use 21