| Description | Product Introduction:This kit is based on spin column adsorption technology and is suitable for recovering 50 bp–30 Kb DNA fragments from agarose gels of various concentrations. In addition, the kit is also suitable for recovering and purifying DNA from PCR products, enzymatic reaction Product Introduction:This kit is based on spin column adsorption technology and is suitable for recovering 50 bp–30 Kb DNA fragments from agarose gels of various concentrations. In addition, the kit is also suitable for recovering and purifying DNA from PCR products, enzymatic reaction solutions, or crude DNA (including genomic DNA) obtained by various methods. Buffer PB contains a pH indicator, and the solution is yellow, which facilitates judging whether the pH value of the solution is suitable for binding to the DNA adsorption column. The DNA recovery efficiency can be as high as 80%, and the purified DNA can be directly used for sequencing, ligation, restriction enzyme digestion (enzyme digestion), PCR, labeling, and other applications.Product Components and Storage Conditions: U1492721 ComponentComponentStorage U1492721ABuffer BL30 mlRT U1492721BBuffer PB25 mlRT U1492721CBuffer DW212 mlRT U1492721DBuffer EB10 mlRT U1492721EFineBind MinElute DNA Spin Columns50个RT U1492721F2 ml Collection Tubes50个RTStorage Conditions:This kit can be stored for 12 months at room temperature (15°C–25°C) under dry conditions. Precipitation may form in Buffer PB at low temperatures; before use, it is necessary to redissolve the buffer in a 37°C water bath and shake it well before use.Precautions:1. The addition of Buffer BL can improve the adsorption capacity of the adsorption column, enhance its uniformity and stability, and eliminate the impact of high temperature/humidity or other adverse environmental factors on the adsorption column. Before use, please check whether Buffer BL is turbid. If turbidity occurs, heat it in a 37°C water bath for a few minutes to restore clarity.2. Buffer PB contains a pH indicator and is yellow, indicating a pH ≤ 7.5.Operating Steps:Before use, add absolute ethanol to Buffer DW2. Please refer to the label on the bottle for the volume to be added.I. Recovering DNA Fragments from Agarose Gels1. Column Equilibration Step: Add 500 µl Buffer BL to the adsorption column (FineBind MinElute DNA Spin Columns) placed in a collection tube. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. (Please use columns processed on the same day)2. Cut the single target DNA band from the agarose gel (remove excess parts as much as possible) and place it in a clean centrifuge tube, then weigh it.3. Add an equal volume of Buffer PB to the gel block (if the gel weighs 0.1 g, its volume can be regarded as 100 µl, so add 100 µl Buffer PB). Incubate in a 50°C water bath for about 10 minutes, gently inverting the centrifuge tube up and down continuously during this period to ensure the gel block is fully dissolved. (If the volume of the gel block is too large, it can be cut into small pieces in advance.)Note: For recovering small fragments < 150 bp, the volume of Buffer PB can be increased to 3 times to improve the recovery rate; after the gel block is completely dissolved, it is best to cool the solution to room temperature before loading onto the column, because the adsorption column has a stronger ability to bind DNA at room temperature. The gel should appear yellow after complete dissolution, and then subsequent operations can be performed. If the color of the solution is orange-red or purple after the gel is completely dissolved, use 10 µl of 3M sodium acetate (pH 5.0) to adjust the color of the solution to yellow before proceeding with subsequent operations. (Buffer PB contains a pH indicator. When pH ≤ 7.5, the solution is yellow, and DNA can effectively bind to the membrane. When the pH is high, the solution turns orange-red or purple and needs to be adjusted.)4. Add the solution obtained in the previous step to the adsorption column (placed in the collection tube), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: The capacity of the adsorption column is 700 µl. If the sample volume is larger than 700 µl, it can be added in batches.5. Add 500 µl Buffer DW2 (with absolute ethanol added before use) to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.6. Repeat step 5.7. Centrifuge at 12,000 rpm for 3 minutes.8. Place the adsorption column into a clean centrifuge tube, 悬空滴加 an appropriate amount of Buffer EB (Buffer EB is heated at 65°C for 3-5 minutes before use, preheated in advance) to the middle part of the adsorption membrane, and let it stand at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.Note: The volume of the eluent should not be less than 30 µl; a smaller volume will affect the recovery efficiency. If the downstream experiment is sensitive to pH, sterile water can be used for elution. The pH of the eluent has a great impact on the elution efficiency. If water is used as the eluent, ensure its pH is within 7.0-8.5 (NaOH can be used to adjust the pH of water to this range). The elution efficiency is low when the pH is below 7.0.II. Recovering DNA from PCR Reaction Solutions or Restriction Enzyme Digestion Solutions1. Column Equilibration Step: Add 500 µl of Buffer BL to the adsorption column (FineBind MinElute DNA Spin Columns) placed in a collection tube. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. (Please use columns processed on the same day.)2. Calculate the volume of the PCR reaction solution or restriction enzyme digestion solution, add an equal volume of Buffer PB to it, and mix thoroughly (there is no need to remove paraffin oil or mineral oil).Note: For recovering small fragments < 150 bp, the volume of Buffer PB can be increased to 3 times to improve the recovery rate; after mixing, the solution should appear yellow before proceeding with subsequent operations. If the solution is orange-red or purple, use 10 µl of 3M sodium acetate (pH 5.0) to adjust the color of the solution to yellow before continuing.3. Add the solution obtained in the previous step to the adsorption column (placed in the collection tube), let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Note: The capacity of the adsorption column is 700 µl. If the sample volume exceeds 700 µl, add it in batches.4. Add 500 µl of Buffer DW2 (ensure absolute ethanol is added before use) to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.5. Repeat step 4 once.6.Centrifuge at 12,000 rpm for 3 minutes.7. Transfer the adsorption column to a clean centrifuge tube, suspend and add an appropriate amount of Buffer EB (preheat Buffer EB by heating at 65°C for 3–5 minutes before use) to the middle of the adsorption membrane, and let it stand at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.Note: The volume of the eluent should not be less than 30 µl; a smaller volume will reduce recovery efficiency. If the downstream experiment is sensitive to pH, sterile water can be used as the eluent. The pH of the eluent has a significant impact on elution efficiency. If water is used as the eluent, ensure its pH is within the range of 7.0–8.5 (NaOH can be used to adjust the pH of water to this range); elution efficiency will be low if the pH is below 7.0... Read More | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | Inquire | Product content:E665636Component50 TStorageE665636ABuffer P115 mLRTE665636BBuffer P215 mLRTE665636CBuffer E315 mLRTE665636DBuffer PS15 mLRTE665636EBuffer PW (concentrate)10 mLRTE665636FEndo-Free Buffer EB10 mLRTE665636GRNase A (10 mg/mL)150 µLRTE665636HEndo-Remover FMwith Collection Tubes50 Product content:E665636Component50 TStorageE665636ABuffer P115 mLRTE665636BBuffer P215 mLRTE665636CBuffer E315 mLRTE665636DBuffer PS15 mLRTE665636EBuffer PW (concentrate)10 mLRTE665636FEndo-Free Buffer EB10 mLRTE665636GRNase A (10 mg/mL)150 µLRTE665636HEndo-Remover FMwith Collection Tubes50 EARTE665636ISpin Columns DMwith Collection Tubes50 EART Product 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 simple, fast, and efficient new method for extracting endotoxin free plasmids. The extracted plasmids remove endotoxins to the maximum extent possible and can effectively remove contamination of genomic DNA, RNA, proteins, etc. The operation is simple and convenient. This reagent kit is suitable for extracting 1-5mL of bacterial solution. On the basis of alkaline lysis of cells, it efficiently and specifically binds plasmid DNA through a new silicon-based membrane. Each adsorption column can adsorb up to 40% µ The plasmid DNA of g is effectively removed using a special buffer system and endotoxin removal filter column, effectively removing impurities such as endotoxins and proteins. The plasmid obtained from this kit has high purity and stable quality, making it particularly suitable for cell transfection. It can also be used for downstream experiments such as DNA sequencing, PCR, PCR based mutations, in vitro transcription, transformed bacteria, and endonuclease digestion.Self prepared reagents: anhydrous ethanol, isopropanol.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 the first use, add all RNase A solution to Buffer P1, mix well, and store at 2-8 ℃. Before use, let it sit at room temperature for a period of time. After returning to room temperature, use.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. Be careful not to come into direct contact with Buffer P2 and Buffer E3, and immediately cover them tightly after use.6.The amount and purity of plasmid extraction are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.Operation steps：1. Take 1-5 mL of overnight cultured bacterial solution and add it to a centrifuge tube (provided). Centrifuge at 13000 rpm (~16200 × g) for 30 seconds to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 250 to the centrifuge tube containing bacterial sediment µ L Buffer P1 (please check if RNase A has been added first), mix thoroughly with a pipette or vortex oscillator, and suspend bacterial precipitation.Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity.3. Add 250 to the centrifuge tube µ L Buffer P2, gently invert and mix 8-10 times, allowing the bacterial cells to fully lyse. Leave 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 250 to the centrifuge tube µ L Buffer E3, immediately invert and mix 8-10 times until white flocculent precipitates appear. Let it stand at room temperature for 5 minutes. Centrifuge at 13000 rpm for 5 minutes, extract the supernatant, and add it to a filter column (Endo Remove FM). Centrifuge at 13000 rpm for 1 minute to filter, and collect the filtrate in a centrifuge tube (self provided).Attention: After adding Buffer E3, it should be mixed evenly immediately to avoid local precipitation. 5. Add 225 to the filtrate µ Mix L isopropanol upside down.6. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ L Buffer PS, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Transfer the mixed solution of filtrate and isopropanol from step 5 to an equilibrium adsorption column (already loaded into a collection tube).8.13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Attention: The maximum volume of the adsorption column is 750 µ L. If the sample volume is greater than 750 µ L can be added in batches. 9. Add 750 to the adsorption column µ L Buffer PW (please check if anhydrous ethanol has been added first), centrifuge at 13000 rpm for 1 minute, and discard the waste liquid in the collection tube.10. Place the adsorption column back into the recovery manifold and centrifuge at 13000 rpm for 1 minute. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column in a new collection tube and add 50-100 to the middle of the adsorption membrane µ L Endo Free Buffer EB, let it stand at room temperature for 2-5 minutes, centrifuge at 13000 rpm for 2 minutes, and collect the plasmid solution into a centrifuge tube- Store the plasmid at 20 ℃.Note: 1) To increase the efficiency of plasmid recovery, the obtained solution can be added back to the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 13000 rpm for 2 minutes, and collected 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... Read More | Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the action of some factors through the regulation of intracellular genes and their products. Cell necrosis is a cell death process that is caused by strong physical and chemical or biological factors to cause disordered changes in cells. The difference between apoptosis and necrosis lies in the characteristic morphological and biochemical changes, including the changes of cell membrane permeability and nuclear chromatin, the contraction of cytoplasm and the loss of membrane asymmetry. The oxazole yellow/pi membrane permeability apoptosis detection kit produced by our company is a dual fluorescence detection kit based on oxazole yellow and PI dyes. This kit is suitable for fluorescence microscopy, flow cytometry, fluorescence microplate reader and other fluorescence detection systems. Oxazole yellow is a non cell membrane penetrating cyanine monomer green fluorescent dye with high affinity for DNA. It basically has no fluorescence when it is not bound to DNA, but can emit bright green fluorescence after binding to DNA. When apoptosis occurs, the permeability of cell membrane changes. At this time, oxazole yellow can enter the cell and bind to DNA, emitting bright green fluorescence. Therefore, it is often used for the detection of apoptosis. It should be noted that oxazole yellow can also stain dead cells, so it needs to be double stained with PI that specifically fluorescently stains dead cells to effectively determine apoptosis. PI (propidium iodide) is a red fluorescent dye that can stain DNA. It is an analog of pyridine bromide that releases red fluorescence after embedding double stranded DNA. Although PI cannot pass through the membrane of living cells, it can cross the damaged cell membrane of dead cells to stain nuclei. Therefore, oxazole yellow combined with PI can be directly used for the detection of apoptosis. Apoptotic cells show green fluorescence, dead cells show both red and green fluorescence positive, and living cells have little or no fluorescence.Components: Components O598364-50T A. Oxazole yellow dye 50 µL B. Propidium Iodide (PI) 50 µLUsage (using flow cytometry as an example):1. Cell preparation(1) For adherent cells, after trypsin digestion, resuspend in culture medium and wash once with pre cooled PBS; The digestion time of trypsin should not be too long to prevent false positives. Note: Digest with trypsin and allow the cells to recover in the optimal cell culture conditions and medium for about 30 minutes, then stain.(2) For suspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash once with pre cooled PBS.2. Cell stainingSuspend cells in pre cooled PBS, with a recommended cell count of 106 cells/mL per sample. Add 1 µ L Oxazole Yellow and 1 µ L to 1 mL of the samplePI， Gently blow and mix well. Incubate on ice in the dark for 30 minutes. Note: We suggest adding the following two experimental controls:Blank tube: negative control group cells, without dye, used to regulate voltage.Single staining tube: Positive control group cells were treated with only two tubes, Oxazole yellow and PI, for regulating compensation.3. Flow detectionAfter incubation, the sample can be directly detected by flow cytometry, or centrifuged at 1000 rpm for 5 minutes, the supernatant can be aspirated, and the sample can be resuspended in 1 mL of pre cooled PBS for flow cytometry detection. Oxazole yellow can be excited by a 488 nm laser, and the detected fluorescence emission spectrum is around 530 ± 30 nm (FITC channel), while the PI channel emission spectrum is around 617 nm (PI or PE channel).Product parameters：Oxazole yellow dye:ex/em = 491 / 509 nm (bound DNA); Propidium iodine:ex/em = 535 / 617 nm (combined with DMatters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 3. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Membrane permeability apoptosis assay... 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