Endo-Free BAC/PAC Plasmid Maxi Isolation Kit(Solution)

RNase A is supplied in ready-to-use glycerol buffer. It can be transported at ambient temperature. Upon receipt，store at room temperature (below 25 °C) for at least 6 months，at 4 °C for 12 months，and at -20 °C for long-term storage. For first use，add the entire supplied RNase A into Solution P1 to reach a final concentration of 100 μg/mL，then store the mixture at 4 °C. If RNase A in Solution P1 becomes inactive，trace RNA contamination may exist in extracted plasmids. Simply replenish RNase A into Solution P1 to resolve this issue. Endotoxin remover is transportable at ambient temperature. It can be stored at 4 °C for 12 months and at -20 °C for long-term preservation.When the ambient temperature is low，SDS in Solution P2 may precipitate and cause turbidity or sediment. Warm the solution in a 37 °C water bath for several minutes until it turns clear. Do not shake vigorously to avoid excessive foaming. Prevent prolonged exposure of reagents to air，which may lead to volatilization，oxidation and pH shift. Tighten the caps immediately after use. Plasmid yield is affected by bacterial culture density，plasmid copy number and other factors. For low-copy plasmids or large plasmids larger than 10 kb，increase the dosage of bacterial pellet，and proportionally raise the volume of Solution P1，P2 and PⅢ. Handle gently during the extraction of large plasmids. Use pipette tips with widened orifices to prevent DNA damage caused by mechanical shearing. No obvious pellet may be visible after centrifugation of DNA precipitation solution. If you worry about DNA loss，retain the supernatant and perform agarose gel electrophoresis after all procedures to verify the final product. Hundreds of micrograms of DNA often adhere to the tube wall and do not form a visible bulk pellet. The concentration and purity of obtained plasmid DNA can be determined by agarose gel electrophoresis and UV spectrophotometry. An OD260 reading of 1 corresponds to approximately 50 μg/mL DNA. Electrophoresis may show one，two or multiple bands，resulting from different migration rates of plasmids with varied supercoiled conformations，which is related to culture duration and operation intensity during extraction. With standard operating procedures，the supercoiled fraction of our product can exceed 95%. To determine the exact molecular size of plasmid DNA，linearize the plasmid via restriction digestion and compare it with DNA molecular weight markers. Circular or supercoiled plasmids have unstable migration positions，so their exact size cannot be identified merely by electrophoresis. This kit adopts the alkaline lysis method to extract plasmid DNA from cultured bacteria. With proprietary solution formulations and endotoxin removal reagents，impurities such as proteins，polysaccharides，endotoxins and RNA can be removed via several simple centrifugation steps to obtain high-purity plasmid DNA. The OD260/280 ratio of purified DNA is generally around 1. 8. The resulting plasmids are directly applicable to experiments with stringent purity requirements，including cell transfection and in vivo animal assays. All post-purification operations are performed in 1. 5 mL microcentrifuge tubes. The protocol is simple without the need for specialized equipment，column purification or phenol-chloroform extraction. Plasmids released from bacterial lysis can be almost fully recovered with negligible DNA loss. This method causes minimal damage to plasmids. It enables efficient purification of large plasmids over 10 kb，even ultra-large BAC/PAC plasmids，as long as they are extractable by alkaline lysis. Plasmids can be eluted in any small volume to reach a concentration up to 5 μg/μL. The supercoiled content is as high as 95% with zero endotoxin contamination，ensuring excellent transfection efficiency. Product Features: No toxic reagents such as phenol and chloroform，nor ethanol precipitation are required. The procedure is rapid and convenient. From 100-140 mL of E. coli LB (Luria-Bertani) culture medium，0. 2-0. 5 mg of pure high-copy plasmid DNA can be rapidly extracted with a recovery rate of 80-90%. The harvested plasmids feature high yield，up to 95% supercoiled fraction and superior purity，which can be directly used for restriction enzyme digestion，transformation，PCR，in vitro transcription，DNA sequencing and other molecular biology experiments. The endotoxin level is extremely low (<0. 1 EU/μg DNA)，making the plasmids suitable for direct cell transfection. Application Scope: For large-scale preparation of high-purity or transfection-grade plasmids，as well as purification of large BAC/PAC plasmids.