Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions
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1 Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) / Fax: (650) mbi@maximbio.com
2 INTRODUCTION Maxim s Plasmid DNA Isolation Column Kits provide a means to isolate plasmid DNA from bacteria. This kit provides all the necessary reagents and protocols to extract high quality plasmid DNA. This general purpose kit can be used to isolate any plasmid DNA but works most efficiently when the plasmid DNA is less than 20 Kb in size. The purified plasmid DNA can be used directly for DNA sequencing and other standard molecular biology techniques. Purification Using Maxim Spin Columns Maxim spin columns will fit into most standard microcentrifuge tubes. In addition, 2 ml collection tubes have been provided along with the kit for the loading and wash steps. DNA can be eluted into 1.5 ml microcentrifuge tubes, which are not provided. Purification procedures using spin columns are intended to extract nucleic acid with no sample to sample cross contamination and ensure the safety of the user while handling samples that are potentially infectious. Elution of Purified Nucleic Acids Purified DNA is eluted into distilled deionized water. To store purified DNA, elution into buffer comprised of 1 mm Tris-Cl, 0.05 mm EDTA, ph 8.3 is recommended. Store at -20 C. Nucleic acid stored in water may degrade due to acid hydrolysis. Determination of Concentration, Yield, and Purity DNA yield is determined from the concentration of eluted DNA in sterile double distilled water as measured by absorbance at 260 nm. Absorbance readings at 260nm should range from The sample should be diluted according to concentration. If the eluted sample contains ng of DNA/µL, should not be diluted with more than 4 volumes of buffer or water. Elution buffer or water should be used to dilute samples and to calibrate the spectrometer. Absorbance readings need to be measured at both 260 nm and 280 nm to determine the concentration of the DNA content of your product. To measure the concentration of DNA alone, use of a fluorimeter is recommended instead. To determine the purity of the sample, calculate the ratio between absorbance at 260 nm to the absorbance at 280 nm. Pure DNA will have an A260/A280 ration from DNA purified using Maxim plasmid DNA isolation column kits will be free from protein and other contaminants. Determination of DNA Size The size of the purified DNA product can be determined by gel electrophoresis. Run the purified product on a 1% agarose gel next to a DNA molecular weight marker. Load µg of DNA per well. Immerse the agarose gel in a 1x TBE electrophoresis buffer and run at 100 volts for 30 min. or until the loading dye front reaches 2/3 of the gel. 2
3 Preparation of bacteria culture 1. Inoculate a single bacterial colony from a fresh Luria-Bertani (LB) agar plate (containing antibiotic) to 1-10 ml LB broth (containing the same antibiotic). 2. Incubate overnight (12-16 hours) at 37 o C in a shaking incubator (250 rpm). For High Copy Number Plasmids - we do not recommend processing more than 5 ml of bacteria culture. If more than 5 ml of bacteria culture is used, the capacity of Maxim s spin column will be exceeded. The yield of plasmids DNA will not increase. For Low Copy Number Plasmids - the 10 ml bacteria culture is recommended for recovery of sufficient plasmid DNA. Proceeding more than 10 ml of bacteria culture will lead incomplete lysing the bacteria. It will increase the contaminants in the plasmid DNA. Handling of Maxim Spin Columns Use care when applying sample or solution to spin column. Try not to allow sample to touch the rim of the column. Change pipet tips between all material transfers. Use aerosol barrier pipet tips whenever possible. Do not let the pipet tip touch the Maxim spin column membrane. Briefly centrifuge 1.5mL microcentrifuge tubes after vortexing. Wear gloves when conducting purification procedure and avoid contact between sample and gloves. Changes gloves immediately should any contact occur. Microcentrifuge protocol Make sure to close the cap of the microcentrifuge tube over the spin column lid before centrifugation. Discard filtrate and the collection tube after centrifugation. Dispose of the filtrate appropriately as it may contain hazardous waste. Open only one microcentrifuge tube containing a spin column at a time, taking care to avoid aerosols. For increased efficiency, use a collection tube rack when conducting purification involving multiple samples. Centrifugation All centrifugation should be conducted at room temperature. The recommended speed for centrifugation is at least 10,000xg. A higher speed will not affect yields and is highly recommended, but lower speeds may be insufficient for transferring solutions completely through the membrane. 3
4 Plasmid DNA Isolation Column Kit Components Catalog No. SA SA Reaction No Cell Resuspension Solution 15 ml 30 ml Cell Lysis Solution 15 ml 30 ml Cell Neutralization Solution 20 ml 40 ml Washing Buffer 30 ml 60 ml Elution Buffer 10 ml 10 ml 1.5 ml microcentrifuge tube Spin Column Sample Source & Size Sample Source: Bacteria Culture Sample Size: 1-10 ml Storage Conditions For optimal results, Maxim spin columns should be stored under dry conditions at room temperature (15-25 C) for a maximum of one year. All of the solutions should be stored at C and are stable for up to 12 months. Required Equipment and Reagents 1.5mL microcentrifuge tubes Sterile DNase-free pipet tips with aerosol barrier Microcentrifuge with rotor for 2mL tubes Product Use Limitations Maxim Plasmid DNA Isolation Column Kits are intended for general nucleic acid purification purposes and not for any specific organism or for any specific clinical purpose. It is up to the user s discretion whether or not Maxim Plasmid DNA Isolation Column Kits are suitable for their specific experimental needs. Products should be handled with care. Please keep the NIH guidelines involving recombinant DNA experiments in mind when using Maxim Column Purification Kits. Product Warranty and Satisfaction Guarantee. Maxim guarantees the quality and performance of our products according to the standards detailed in our instruction manuals and publications. It is up to the user s discretion whether or not Maxim Plasmid DNA Isolation Column Kits are suitable for their specific experimental needs. If the Maxim Plasmid DNA Isolation Column Kit has been used properly yet fails to perform to your satisfaction, then Maxim will replace the kit free of charge or refund the purchase price for the item. To return or exchange an item, simply call our Technical Service Department. The Maxim technical service information is listed on the last page. Maxim reserves the right to modify or change any of its products to enhance performance. 4
5 Technical Assistance Maxim s Technical Service department is staffed by experienced scientists with expertise in various fields of immunology, pathology, and molecular biology, etc. If you have any questions of Maxim s products, please contact us at our technical hot line ext 14 or to mbi@maximbio.com. 5
6 PROTOCOL 1. Harvest 5-10 ml bacterial culture by centrifugation for 5 minutes at 10,000 x g in a tabletop centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media. 2. Add 250 µl of Cell Resuspension Solution and completely resuspend the bacterial pellet by vortexing or pipetting. Then, transfer the resuspended bacteria to a sterile 1.5 ml microcentrifuge tube. Note: It is very important to thoroughly resuspend the bacteria pellet. 3. Add 250 µl of Cell Lysis Solution and mix well by inverting several times until the lysate clears. Note: Do not vortex in this step. It is very important to observe the clearing of the lysate. 4. Add 350 µl of Cell Neutralization Solution and immediately mix well by inverting several times and Centrifuge for 5 minute at maximum speed (around 14,000 x g) at room temperature. Note: Do not vortex in this step. 5. Carefully transfer the clear lysate (approximately 850 µl) to the spin column in a 2.0 ml microcentrifuge tube provided and centrifuge for 1 minute at maximum speed. Note: Avoid disturbing or transferring any of the white precipitate with the supernatant. 6. Carefully transfer the spin column into a fresh 2.0 ml microcentrifuge tube provided. Add 500µL Washing Buffer into the spin column and centrifuge for 1 minute at maximum speed. 7. Carefully transfer the spin column into a new 1.5 ml microcentrifuge tube (not provided) and centrifuge for 1 minute at maximum speed. Note: This avoids any residual washing buffer. 8. Carefully transfer the spin column into a new 1.5 ml microcentrifuge tube (not provided). Add 100 µl Elution Buffer to the spin column, let the column stand for 1 minute, and centrifuge for 1 minute at maximum speed. Note: Eluted plasmid DNA is stable if kept at -20 C for long term storage or at 4-8 C for short term storage. 6
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