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超滤管[15ml 10KD],装
装有 Ultracel-10 超
Millex 33mm Durapore
Amicon Ultra-0.5 mL 
Immobilon-P转印膜Contents
Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBindTM matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or elution buffer. Unlike other procedures, our patented plasmid purification kit has no guanidine salt in the buffer, the purified DNA is guanidine/ion exchange resin residues free which enable the high performance of downstream applications such as transfection, restriction mapping, library screening, sequencing, as well as gene therapy and genetic vaccinations. Important Notes Plasmid Copy Numbers: The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid. The protocols are optimized for high copy number plasmid purification. For low copy number plasmids, both the culture volume and the buffer volume need to be scaled up 2 to 3 times. Reference Table 1 for the commonly used plasmids, Table 1 Commonly used plasmids.
Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10 and DH5a yield high-quality plasmid DNA. endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis. We recommend transform plasmid to an endA- strain if the yield is not satisfactory. For purifying plasmid DNA from endA+ strains (Table 2), we recommend use product PD1714. Table2 endA strains of E. Coli.
Optimal Cell Mass (OD600 x mL of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to a density of OD600 2.0 to 3.0. If rich medium such as TB or 2xYT are used, make sure the cell density doesn’t exceed 3.0 (OD600). A high ratio of biomass over lysis buffers result in low DNA yield and purity. Culture Volume: Use a flask or tube with a volume at 4 times the culture medium to secure optimal condition for bacteria growth. Don’t exceed the maximum culture volume suggested in the protocol. Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity. Table 3 The optimal cell mass, culture Volume and Binding Capacity for the mega DNA units,
Storage and Stability Buffer A1 should be stored at 4°C once RNase A is added. All other materials can be stored at room temperature (22-25oC). The Guaranteed shelf life is 12 months from the date of purchase. Before StartingPrepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps. Important: · RNase A: Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use. · Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50°C to dissolve the precipitates before use. · Buffer C1 may form precipitates below 10°C, warm up at 37°C to dissolve the precipitates before use. · Keep the cap tightly closed for Buffer B1 after use. · The proper volume of buffer ratio of A1:B1:C1: 100% ethanol =1:1:1.2:1.2. · Make sure the availability of centrifuge and vacuum manifold, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by vacuum. Materials supplied by users: · 70% ethanol and 100% ethanol. · Pump-driven vacuum system, 500 mL bottle or 1,000 mL bottle (Corning# 430518 or 430282) or equivalent pyrex glass bottles. · 50 mL conical tubes. Kit Contents
Safety InformationBuffer C1 contains acetic acid, use gloves and protective eyewear when handling. EZgeneTM Plasmid ezFilter Megaprep 3 Protocol 1. Inoculate 500 mL LB containing appropriate antibiotic with 500 µL fresh starter culture. Grow at 37°C for 14-16 h with vigorous shaking. Note: The best way to prepare a starter culture: Inoculate a single colony from a freshly grown selective plate into 1 mL LB medium containing the appropriate antibiotic and grow at 37°C for 6-8 h with vigorous shaking (~250 rpm). The buffer volumes need to be scaled up if processing over 500 mL of culture. Note: Do not use a starter culture that has been stored at 4°C. Note: Do not grow starter culture directly from glycerol stock. 2. Harvest 500 mL overnight bacterial cells by centrifugation at 5,000 x g for 10 minutes at room temperature. Decant or aspirate medium and discard. Note: Remove the residual medium completely for optimal cell lysis and neutralization. 3. Resuspend the bacterial pellet in 30 mL Buffer A1 (Add RNase A to Buffer A1 before use). Pipet or vortex till the bacterial pellet dispersed thoroughly (Complete resuspension is critical for optimal yields). 4. Add 30 mL Buffer B1, mix gently but thoroughly by inverting 10 times and incubate at room temperature for 5 minutes to obtain a cleared lysate. Note: Do not incubate longer than 5 minutes. Over-incubating causes genomic DNA contamination and plasmid damage. Avoid vigorous mixing as this will shear the genomic DNA. 5. Add 8 mL Buffer C1 and mix immediately by inverting 5 times and sharp hand shaking for 10 times till a flocculent white precipitate forms. Note: It is critical to mix the lysate well. If the mixture still appears conglobated, brownish or viscous, more mix is required to completely neutralize the solution. 6. Incubate the mixture at room temperature for 10 minutes. Add 28 mL Buffer C1 to the mixture, mix by inverting 2 times and set for 2 minutes. 7. Attach the 2-layer filter unit to a sterile 500 mL or 1000 mL standard bottle (Corning# 430518 or 430282 or equivalent pyrex glass bottle) and screw tight. Connect the unit to a pump-driven vacuum system. 8. Transfer the clear lysate from the bottom of the mixture (use a 50 mL serological pipet) to the filter unit. Stand by for 5 minutes and turn on the vacuum with low vacuum force and increase to maximum vacuum force after 5 minutes. Figure 1. Instruction of filter assembling. Note 1: Low vacuum force prevents clogging of the filter membranes. Note 2: Use a 50 mL serological pipet to transfer the relatively clear lysate from the bottom of the lysate bottle to the filter unit. This will speed up the flow rate of the filter unit. Normally around 80 mL lysate can be filtered through the filter unit within 10-15 minutes. Pour the remaining white precipitates to the filter unit when most of the lysate has been filtered through. Note 3: If the flow through gets too slow, turn off the vacuum and wait for 1 minute. Carefully detach the upper filter cup and replace it with the replacement cup. Assemble the unit as Figure 1. Pour the lysate from the original cup to the replacement cup. Turn on the vacuum and filter the rest of the lysate. 9. When most of the lysate has been filtered through the unit, turn off the vacuum, wait for 1 minute, detach the unit and discard the upper filter cup including the rubber rings. Note: The DNA is in the collection bottle. 10. Connect the DNA unit to a 500 mL or 1,000 mL standard bottle and screw tight. Connect the DNA unit to the vacuum with the vacuum off. Add 36 mL 100% ethanol to the lysate bottle. Mix well by sharp hand shaking 3-5 times and immediately pour half of the lysate/ethanol mixture to the DNA unit and turn on the vacuum. 11. Pour the rest of the lysate/ethanol mixture into the DNA unit. When all the lysate pass through the DNA unit, vacuum for 1 minute. 12. Wash the DNA membrane with 50 mL 70% ethanol and vacuum for 1 minute at maximum force. Repeat this step once. 13. Add 50 mL 100% ethanol evenly to the DNA membrane and vacuum for 1 minute. Turn off the vacuum, wait for 1 minute, and discard the liquid waste in the bottle. Reconnect the bottle to the DNA binding unit. Turn on the vacuum for 20 minutes at maximum force to remove the ethanol residues. Note: Residual ethanol can be removed more efficiently with the column lid open. It is critical to remove residual ethanol completely. 14. Turn off the vacuum, wait for 1 minute, and replace the 500 mL or 1,000 mL bottle with a sterile 50 mL conical tube, screw tight. 15. Add 8 mL sterile ddH2O or Elution Buffer evenly to the membrane and incubate for 2 minutes. Turn on vacuum to elute DNA. Typically, 3-5 mL of DNA containing solution can be collected. This is the 1st elution. 16. Turn off the vacuum and replace the 50 mL conical tube with another sterile 50 mL conical tube, screw tight. Add 5 mL Elution Buffer and incubate for 1 minute. Turn on the vacuum and collect the 2nd elution, typically 3 mL of solution can be collected. Note: If ddH2O is used for eluting DNA, make sure the pH is ≥ 7.0. Note: The DNA is ready for downstream applications such as cloning/subcloning, RFLP, Library screening, in vitro translation, sequencing, transfection of robust cell lines (HEK293 cells). Note: It’s highly recommended to remove the endotoxin (PD1615) if the DNA is used for endotoxin-sensitive cell lines, primary cultured cells or microinjection. Note: Two elutions give rise to maximum DNA yield. For maximum yield and higher concentration, pool the elutions together, add 0.1 volume 3M Potassium Acetate or Sodium Acetate (pH 5.2) and 0.7 volume isopropanol. Centrifuge at top speed for 10 min. Discard supernatant. Wash the DNA with 1000 µL 70% ethanol, centrifuge for 5 min, carefully decant. Air-dry the pellet for 10-20 minutes in a tissue culture hood. Resuspend the DNA in Elution Buffer or sterile ddH2O. DNA concentration (µg/mL) = OD260 nm x 50 x dilution factor. Purification of Low-Copy-Number Plasmid and CosmidThe yield of low copy number plasmid is normally around 0.1 – 1 µg /mL of overnight culture. For isolating low copy number or medium copy number plasmid DNA, use the following guideline: 1. Culture volume: Use 2 x volumes of the high copy number culture 2. Use 2 x volumes of the Buffer A1, Buffer B1 and Buffer C1 and 100% ethanol. Additional buffers can be purchased from Biomiga. 3. Use same volume of Wash Buffer (70% ethanol and 100% ethanol) and Elution Buffer. Trouble Shooting Guide
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Limited Use and WarrantyThis product is warranted to perform as described in its labeling and in Biomiga’s literature when used in accordance with instructions. No other warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Biomiga. Biomiga’s sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of Biomiga, to replace the products, Biomiga shall have no liability for any direct, indirect, consequential, or incidental damage arising out of the use, the results of use, or the inability to use it product. For technical support or learn more product information, please visit our website at www.biomiga.com or contact us at (858) 603-3219. *FOR RESEARCH USE ONLY. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
