Description This kit uses unique silica membrane adsorption technology to efficiently and precisely bind plasmid DNA. Meanwhile, the unique solution ER can effectively remove endotoxin; the extraction process only takes 1 hour, which is convenient and fast. The following procedure is suitable for the extraction of 1-5 ml of overnight cultured E. coli. The ratio and quality of plasmid extraction are related to the host bacteria species and culture conditions, cell lysis, plasmid copy number, plasmid stability, antibiotics, and other factors. The plasmid extracted by this kit can be used to transfect cultured cell lines and for routine operations, including restriction enzyme digestion, PCR, sequencing, ligation, and other experiments. Kit Contents Contents GDP123-02 (50 preps) GDP123-03 (200 preps) Buffer P1 15 ml 60 ml Buffer P2 15 ml 60 ml Buffer P4 15 ml 60 ml Endotoxin Removal Buffer ER 5 ml 20 ml Buffer ED 35 ml 140 ml Buffer PW 15 ml 2 × 30 ml Buffer TB 15 ml 30 ml RNase A (10 mg/ml) 150 μl 600 μl Spin Columns CP4 50 200 Collection Tubes 2 ml 50 200 RNase-Free Centrifuge Tubes (1.5 ml) 50 200 Required Reagents 96-100% ethanol Yield For Reference Plasmid type Bacterial fluid volume Yield Plasmid Low copy 1–5 ml 3–12 μg pBR322, pACYC and its derivative vector pSC101 and its derivative vector, SuperCos, pWE15 High copy 1–5 ml 6–30 μg pTZ, pUC, pBS, pGM-T Storage The kit can be stored dry at room temperature (15-30°C) for up to 15 months without showing any reduction in performance and quality. If any precipitate forms in the buffers, it should be dissolved by warming the buffers at 37°C before use. RNase A (10 mg/ml) can be stored for 15 months at room temperature (15-30°C). After addition of RNase A, Buffer P1 is stable for 6 months at 2-8°C. Important Notes Add the provided RNase A solution to Buffer P1 before use (use 1 vial RNase A per bottle Buffer P1), mix, and store at 2-8°C. 100% ethanol should be added to the buffer PW before the first use according to the instructions on the label of the reagent bottle. Check whether the buffer P2 and P4 before use for salt precipitation. If necessary, dissolve the buffer by warming at 37°C for several minutes. Be careful not to mix buffer P2 and P4 directly, and tighten the lid immediately after use. All centrifugation steps are carried out at 12,000 rpm (~13,400 ×g) in a bench-top microcentrifuge at room temperature (15-30°C). The amount of plasmid extracted is related to the concentration of bacterial culture, plasmid copy number and other factors. If the proposed plasmid is a low-copy plasmid or a large plasmid larger than 10 kb, the amount of bacteria used should be increased, and the amount of P1, P2 and P4 should be increased proportionally, and the elution buffer should be preheated at 65-70°C. The adsorption and elution times can be extended appropriately to increase the extraction efficiency. Plasmid DNA Concentration and Purity Test The plasmid DNA obtained can be tested for concentration and purity by agarose gel electrophoresis and UV spectrophotometer. The electrophoresis may be a single band or 2 to 3 DNA bands, which is mainly related to the length of the extract incubation time and the degree of vigorous operation during extraction. An OD260 value of 1 corresponds to approximately 50 μg/ml double-stranded DNA. The OD260/OD280 ratio should be 1.7-1.9. If deionized water is used in the elution instead of elution buffer, the ratio will be low, but it does not indicate a low purity because the pH and the presence of ions will affect the light absorption value. Documents Manual
