| Description | G665573 Component 10 T Storage G665573A Buffer P1 30 mL RT G665573B Buffer P2 30 mL RT G665573C Buffer E3 30 mL RT G665573D Buffer PS 15 mL RT G665573E Buffer PW (concentrate) 10 mL RT G665573F Endo-Free Buffer EB 30 mL RT G665573G RNase A (10 mg/mL) 600 碌L RT G665573H Endo-Remover FX 10 EA G665573 Component 10 T Storage G665573A Buffer P1 30 mL RT G665573B Buffer P2 30 mL RT G665573C Buffer E3 30 mL RT G665573D Buffer PS 15 mL RT G665573E Buffer PW (concentrate) 10 mL RT G665573F Endo-Free Buffer EB 30 mL RT G665573G RNase A (10 mg/mL) 600 碌L RT G665573H Endo-Remover FX 10 EA RT G665573I Plungers 10 EA RT G665573J Spin Columns DX with Collection Tubes 10 EA RT G665573K Centrifuge Tubes (15 mL) 10 EA RTProduct IntroductionThis kit is specially designed for the efficient and rapid extraction of plasmids from 15-50 ml of bacterial fluids. On the basis of cell lysis by alkaline lysis method, it adopts unique silicon matrix membrane adsorption technology to bind plasmid DNA efficiently and exclusively, and each adsorption column can adsorb up to 250 µg of plasmid DNA; at the same time, it adopts a special buffer system and endotoxin removal filter to effectively remove endotoxin, genomic DNA, RNA, protein and other impurities. The plasmids obtained from this kit are of high purity and stable quality, and can be used for cell transfection, as well as DNA sequencing, PCR, in vitro transcription, endonuclease digestion and other experiments.Self-contained reagents: anhydrous ethanol, isopropanol.Pre-experiment Preparation and Important Notes1. All components are stable for 1 year in a dry, room temperature (15-30°C) environment, and longer by placing the adsorption columns at 2-8°C. Buffer P1 with RNase A is stable for 6 months at 2-8°C.2. Before the first use, add all of the RNase A solution to Buffer P1, mix well, and store at 2-8°C. Before use, it needs to be left at room temperature for a period of time, return to room temperature and then use.3. Anhydrous ethanol should be added to Buffer PW before the first use according to the instructions on the reagent bottle label.4. Please check Buffer P2 and Buffer E3 for crystallization or precipitation before use. If there is any crystallization or precipitation, the clarification can be restored by taking a water bath at 37℃ for a few minutes.5. Be careful not to touch Buffer P2 and Buffer E3 directly, and tighten the lid immediately after use.6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors.7. The adsorption columns treated with Buffer PS should be used immediately to avoid leaving them for too long.Operation steps1.Take 15-50 ml of fresh bacterial solution from the overnight culture, add it to a centrifuge tube (self-prepared) and centrifuge at 5000 × g for 10 minutes to collect the bacteria, and aspirate all the supernatant as much as possible.2.Add 2.5 ml of Buffer P1 to the centrifuge tube in which the bacterial precipitate has been left (please check that RNase A has been added first) and suspend the bacterial precipitate by mixing thoroughly using a pipette or vortex shaker. Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect and make the extraction amount and purity low.3.Add 2.5 ml of Buffer P2 to the centrifuge tube, mix gently up and down 8-10 times to fully lyse the organisms, and leave at room temperature for 3-5 minutes. At this point the solution should become clear and viscous. Note: Mix gently, do not shake vigorously, so as not to interrupt the genomic DNA and cause genomic DNA fragments to be mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.4.Add 2.5 ml of Buffer E3 to the centrifuge tube and mix immediately by turning up and down 8-10 times, at which time a white flocculent precipitate appears. Note: Buffer E3 should be mixed immediately after addition to avoid localized precipitation.5.Install the cap of the filter (Endo-Remover FX), transfer the solution obtained in step 4 to the filter, wait until the white flocculent precipitate floats on the upper layer of the solution, remove the cap of the filter, align the filter with a clean 15 ml centrifuge tube (supplied), and slowly push the handle (Plungers) to filter, so that as much as possible of the solution passes through, and the filtrate is collected in the centrifuge tube.6.Add 1/3 solution volume of isopropanol to the filtrate and mix upside down.7.Column Equilibrium: Add 1ml Buffer PS to the adsorption column (Spin Columns DX) that has been loaded into a 15ml centrifuge tube, centrifuge for 2 minutes at 2500 x g. Pour off the waste liquid from the centrifuge tube and put the adsorption column back into the centrifuge tube.8.The mixture of filtrate and isopropanol from step 6 was transferred to the equilibrated adsorption column (which had been loaded into a collection tube).9.Centrifuge at 2500 x g for 1 minute, pour off the waste solution in the collection tube and put the adsorption column back into the collection tube. Note: The maximum volume of the adsorption column is 4 ml, so the solution obtained in step 8 is passed through the column in 2 times.10.Add 2 ml of Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 2500 × g for 1 min, and pour off the waste liquid in the collection tube.11.Repeat step 10.12.The adsorbent column was put back into the collection tube and centrifuged at 2500 × g for 2 min, the waste liquid was poured off, and the column was left to dry at room temperature for 5 min.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.)13. Place the adsorption column in a new 15 ml centrifuge tube, add 0.5-1 ml Endo-Free Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 2500 × g for 2 minutes, and collect the plasmid solution into the centrifuge tube. -20°C to store the plasmid.Note: 1) In order to increase the recovery efficiency of the plasmid, the obtained solution can be reintroduced into the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 2500 x g for 2 minutes, and the plasmid solution can be collected into a centrifuge tube.2) When the plasmid copy number is low or >10kb, Endo-Free Buffer EB can increase the extraction efficiency by preheating at 65-70°C in a water bath... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of plasma membrane to reflect cell viability. The kit can be used for fluorescence microscopy, flow cytometry, microplate reader and other fluorescence detection systems. This kit can be applied to most eukarYOtic mammalian cells, including some tissues with adherent nuclei, but it is not applicable to fungi and yeast. Compared with trypan blue, the kit is faster, safer and more sensitive.Component： Product parameters：Calcein am: ex/em = 494 / 517 nm; Ethd-i: ex/em = 528 / 617 nm (bound DNA)Usage:Fluorescence microscopy detection1. Prepare working fluidPreparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Remove the original solution of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium, vortex well. The above working solution can be directly used for cell staining.Note: The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. The concentration selection of Calcein AM and EthD-I varies depending on the type of cell used, with a recommended concentration range of 0.1-10 µ M.2. Prepare cells and conduct experiments(1) For adherent cells, they can be washed 2-3 times with 1 × PBS before staining. For suspended cells, centrifuge at room temperature of 250-1000 × g for 5 minutes and collect cells for staining.(2) Wash the cells thoroughly 2-3 times with 1 × PBS to remove residual esterase activity.(3) For adherent cells, add sufficient amount of Calcein AM/EthD-I staining solution. For suspended cells, add an appropriate amount of staining solution to control the cell density between 1-5 × 105/mL.(4) Incubate at room temperature in dark for 15-20 minutes (if the working solution concentration is high or the incubation temperature is high, the incubation time should be appropriately reduced).(5) Observe the labeled cells under a fluorescence microscope.Flow cytometry detection1. Remove the reagent and restore it to room temperature.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Take out the original solution of Calcein AM and EthD-I, and restore to room temperature. Add 20 µ L 2 mMEthD-I and 5 µ Vortex mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium. The working fluid can directly stain cells.3. Wash cells thoroughly 2-3 times with 1 × PBS.4. Suspend cells with 0.5 mL of staining solution and control the cell density to 1-5 × 105/mL.Note: It is recommended to prepare two additional cell samples, each containing only one dye (Calcein AM and EthD-I), for compensatory regulation of flow cytometry single staining; Prepare another cell sample containing only buffer solution (which should be consistent with the buffer used to prepare Calcein AM and EthD-I detection working solutions) as a negative control for flow cytometry analysis.5. Incubate at room temperature in dark for 15-20 minutes.6. Within 1-2 hours, cell activity was detected by flow cytometry. Calcein AM can be excited by a 488 nm laser, with fluorescence emission spectra detected at around 530 nm and EthD-I emission spectra at around 610 nm.Note: When using the cell circle gate, attention should be paid to excluding cell debris and using a single staining tube to regulate compensation. Double staining tube flow cytometry should obtain two relatively independent cell populations: a live cell population displaying green fluorescence and a dead cell population displaying red fluorescence.ELISA reader detection1. Cultivate an appropriate amount of adherent or suspended cells in a 96 well black ELISA plate.Note: Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution:Remove the original solutions of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM 10 mL PBS or other serum-free buffer or culture medium, vortex well.Note: (1) 10 mL of staining solution is sufficient to stain a 96 well plate, and the volume of the staining solution can be adjusted according to experimental needs. The concentrations of Calcein AM and EthD-I can range from 0.1 to 10 µ Explore between M.(2) The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. EthD-I working solution can be stored at -20 ℃ for at least one year.3. Wash the cells thoroughly with 1 × PBS to remove residual esterase activity. For adherent cells, add 100 to each well µ Wash cells with PBS. For suspended cells, add 100 µ Resuspend cells with L PBS and centrifuge to remove the supernatant. Repeat the above operation.4. Add 100 to each hole µ L PBS.5. Add 100 to each hole µ L staining solution, making the total volume of each well 200 µ L. The final concentration of Calcein AM is 1 µ M. The final concentration of EthD-I is 2 µ M. Gently shake the culture plate to evenly cover the cells with the liquid.Incubate at room temperature in dark for 30-45 minutes.Note: The optimal incubation time varies for different cells, with 30 minutes as the initial incubation time. Subsequently, the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect.7. Enzyme reader detection. When the ELISA reader is set to fluorescein, it can detect Calcein AM; When the ELISA reader is set to rhodamine or Texas Red, EthD-I can be detected. Select the optimal emission and excitation wavelengths based on spectral characteristics.Note: By comparing the relative fluorescence values (RFU) measured between the sample group and the control group, the changes in the number of dead and live cells can be obtained. Another method of data analysis is also provided below.The following method can calculate the ratio of live cells to dead cells in a certain region. The required samples include dead cell control group, live cell control group, and the sample group to be tested. Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.1. Prepare staining solution and follow the above steps to stain cells. Additionally, prepare 1 mL and 2 mL separately µ M Calcein AM and 4 µ M EthD-I solution, stain the control group according to the following instructions. For the following groups of cells or cell-free groups, it is necessary to maintain complete consistency in cell count, detection of working solution concentration, incubation time, and incubation temperature.2. Measurement of sample group and control group:A. The measured values of the sample group at 645 nm are denoted as Calcein AM and EthD-I=F (645) sam.B. The measured values of the sample group at 530 nm are denoted as Calcein AM and EthD-I=F (530) sam.C. The measurement value of dead cell EthD-I single staining control group at 645 nm is denoted as EthD-I=F (645) maxD. The measurement value of dead cell Calcein AM single staining control group at 645 nm is recorded as Calcein AM=F (645) minE. The measurement value of live cell EthD-I single staining control group at 530 nm is recorded as EthD-I=F (530) min.F. The measurement value of live cell Calcein AM single staining control group at 530 nm is denoted as Calcein AM=F (530) max.G. A blank control well without cells (with or without dye), the detection value at 530 nm is recorded as F (530) 0.H. A blank control well without cells (with or without dye), the detection value at 645 nm is recorded as F (645) 0.3. Calculate the ratio of dead cells to live cells based on measurement data:%Live Cells=(B-E) ÷ (F-E)%Dead Cells=(A-D) ÷ (C-D)Determine the ratio of live cells to dead cells in a certain areaBy creating fluorescence spectral standard curves at 530 nm and 645 nm, the number of dead and live cells can be determined, and the fluorescence intensity of each dye is linearly related to the number of dead or live cells in the sample.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. phenol red or serum may interfere with the detection of this kit. 3. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Dead and live cell staining (animal)... 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