GoldVac EndoFree Plasmid Maxi Kit (C09-1136-174)

Product content： Component G665655 2 preps G665655               10 preps Buffer P1 30 ml 125 ml Buffer P2 30 ml 125 ml Buffer E3 30 ml 125 ml Buffer PS 15 ml 30 ml Buffer PW（concentrate） 10 ml 50 ml Endo-Free Buffer EB 10 ml 30 ml RNase A（10 mg/ml） 0.6 ml 2 ml Plungers 2 10 Endo-Remover FQ 2 10 DNA-Binding Tubes 2 10 VacConnectors 2 10 Centrifuge Tubes（50 ml） 2 20Product Introduction:Endotoxins are a common pollutant in plasmid extraction. Due to the high sensitivity of eukaryotic cells to endotoxins, the presence of endotoxins in plasmids can greatly reduce the transfection efficiency of eukaryotic cells. This reagent kit provides a new method for rapid and easy large-scale plasmid preparation. Each time, 100-300 ml of bacterial solution can be processed to obtain up to 2 mg of transfected plasmid DNA. Multiple samples can be purified simultaneously using a vacuum device, and plasmid extraction can be completed in 45 minutes, effectively reducing manual operation time. A special buffer system and endotoxin removal filter effectively remove impurities such as endotoxins, genomic DNA, RNA, proteins, etc. The plasmid obtained from this kit has high purity and extraction capacity, making it particularly suitable for cell transfection. It can also be used for DNA sequencing, PCR, in vitro transcription, endonuclease digestion and other experiments.Self prepared reagents: anhydrous ethanol, isopropanol, vacuum pump, waste liquid collection device, vacuum purification device.Preparation and important precautions before the experiment:1. All components can be stably stored for 1 year in a dry, room temperature (15-30 ℃) environment. The adsorption column can be stored for a longer time at 2-8 ℃.Buffer P1 with RNase A added can be stably stored for 6 months at 2-8 ℃. 2.Before use, add RNase A to Buffer P1 (add all RNase A provided in the reagent kit), mix well, and store at 2-8 ℃. Before use, it is necessary to leave it at room temperature for a period of time, and then use it after returning to room temperature.3.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in Buffer P2 and Buffer E3. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. Note that Buffer P2 and Buffer E3 contain irritating substances. Please wear gloves when operating and immediately cover the lid after use.6. It is best to use the adsorption column treated with Buffer PS immediately to avoid prolonged storage time that may affect its effectiveness.7. Please prepare the waste liquid collection device/buffer bottle CWE100, vacuum purification device CWE200, and vacuum pump CWE300.Operation steps:1. Take 100-300 ml of overnight cultured bacterial solution and add it to a centrifuge tube (provided). Centrifuge at 12000 × g for 2-3 minutes to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 12 ml of Buffer P1 to the centrifuge tube containing bacterial sediment (please check if RNase A has been added first), mix thoroughly with a pipette or vortex oscillator, and suspend bacterial sediment. Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity.3. Add 12 ml of Buffer P2 to the centrifuge tube, gently invert and mix 8-10 times, allowing the bacterial cells to fully lyse. Let it stand at room temperature for 3-5 minutes. At this point, the solution should become clear and viscous.Attention: Mix gently and do not shake vigorously to avoid interrupting genomic DNA and mixing genomic DNA fragments in the extracted plasmid. If the solution does not become clear, it indicates that the bacterial count may be too high and the lysis may not be complete. The bacterial count should be reduced.4. Add 12 ml of Buffer E3 to the centrifuge tube, immediately invert and mix 8-10 times. At this point, white flocculent precipitates appear, and let it stand at room temperature for 5 minutes. Pour all the solution into the Endo Remove FQ filter, slowly push the plunger to filter, and collect the filtrate in a clean 50 ml centrifuge tube.Attention: 1) After adding Buffer E3, it should be immediately mixed to avoid local precipitation.2) After adding E3, if there is excessive precipitation, centrifuge 12000 × g for 10 minutes, and then pour the supernatant solution into the endotoxin removal filter.5. Add 0.3 times the volume of the filtrate to isopropanol, invert and mix well.Attention: Adding too much isopropanol can easily lead to RNA contamination.6. Connect the negative pressure device correctly, connect the VacConnectors to the DNA binding tubes, and then insert them into the socket of the negative pressure device. Attention: Ensure that the connection between the connecting pipe and the adsorption column is stable to prevent air leakage.7. Column equilibrium: Add 2 ml of Buffer PS to the DNA Binding Tubes, turn on and adjust the negative pressure to -300~-700 mbar, and remove the solution from the column.8. Transfer the mixed solution of filtrate and isopropanol from step 5 to an equilibrium adsorption column and remove the solution on the column.9. Add 10 ml of Buffer PW to the DNA Binding Tubes (please check if anhydrous ethanol has been added first) and remove the solution from the column.10. Repeat step 9.11. Maintain negative pressure suction for 10 minutes to remove residual rinsing solution from the adsorption membrane and dry the adsorption membrane. If more than 6 samples are drawn at once, the negative pressure suction time can be appropriately extended. After the adsorption film is completely dried, turn off the negative pressure switch.Note: 1) The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, 12. PCR, etc.).2) Determine the suction time of the negative pressure device based on the drying condition of the adsorption film. If it is not completely dried, an additional drying step can be added, drying at 65 ℃ for 30 minutes to completely dry the residual solution inside the adsorption film.12. When the pressure is restored to 0 mbar, remove the adsorption column and place the DNA adsorption column in a new 50 ml Centrifuge Tube. Add 1-3 ml Endo Free Buffer EB to the middle of the adsorption membrane and let it stand at room temperature for 2-5 minutes. Centrifuge at 12000 x for 5 minutes and collect the plasmid solution into the centrifuge tube- Store the plasmid at 20 ℃.Note: 1) In order to increase the recovery efficiency of plasmids, the obtained solution can be re added to DNA Binding Tubes, left at room temperature for 2-5 minutes, and centrifuged at 12000 × g for 5 minutes. Collect the plasmid solution into a centrifuge tube.2) When the plasmid copy number is low or>10 kb, preheating the Endo Free Buffer EB in a water bath at 65-70 ℃ can increase the extraction efficiency.