| Description | Inquire | Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (excitationat 490 nm, emission at515 nm). Therefore, Calcein-AM only stains viable cells. On the other hand, PI, a nuclei staining dye, cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (excitation: 535 nm,emmision: 617 nm). Since both calcein and PI-DNA can be excited with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. With 545 nm excitation, only dead cells can be observed (Fig. 1). Since optimal staining conditions differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be individually determined. Please note that PI is suspected to be highly carcinogenic;careful handling is required.Required Equipment and Materials:Microscope with 490 nm excitation filter and 530 nm emission filter；CO2incubator；10 µl and 200 µl adjustable pipettes, PBSSolution A (Calcein-AM)；Solution B (PI) Storage Condition: -20oC ；Shipping Condition: blue ice.Application:Assay Procedure1)Add 2.5 µl Solution A and 12.5 µl Solution B to 5 ml PBS to prepare assay solution.*2)Wash the cell with PBS several times to remove residual esterase activity.3)Add 100uLof assay solution to200uL105~106CELLSsolution and incubate the mixture at 37oC for 15 min.4)Detect fluorescence using a fluorescence mircoscope with 490 nm excitationfor simultaneous monitoring of viable and dead cells.With 545 nm excitation, only dead cells can be observed.*The following steps may be necessary tooptimizethe suitable concentration of each reagent:1)Prepare dead cells by 10 min incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 min incubation in 70% ethanol.2)Stain dead cells with 0.1-10 µM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3)Stain dead cells with 0.1-10 µM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stainviable cells with that Calcein-AM solution to check whether the viable cell can be stained... 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 | Product content:D665967Component200 TStorageD665967ABuffer PB120 mLRTD665967BBuffer PS60 mLRTD665967CBuffer PW (concentrate)25 mLRTD665967DBuffer EB30 mLRTD665967ESpin Columns DM with Collection Tubes200 EART Product Introduction: This reagent kit adopts a new silicon-based membrane technology and Product content:D665967Component200 TStorageD665967ABuffer PB120 mLRTD665967BBuffer PS60 mLRTD665967CBuffer PW (concentrate)25 mLRTD665967DBuffer EB30 mLRTD665967ESpin Columns DM with Collection Tubes200 EART Product Introduction: This reagent kit adopts a new silicon-based membrane technology and reagent formula. Through a rapid and simple three-step process of binding, washing, and elution, 100 bp-10 kb DNA fragments can be purified and recovered from PCR products or enzyme reaction solutions (enzyme cutting, linking, probe labeling, etc.). Each adsorption column can adsorb up to 10 kb of DNA fragments µ G DNA, while minimizing impurities such as primers, oligonucleotides, enzymes, etc. The purified and recovered DNA has high purity and concentration, good integrity, and high recovery rate, and can be directly used for molecular biology experiments such as sequencing, linking and transformation, labeling, and in vitro transcription.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. All components can be stably stored in a dry, room temperature (15-30 ℃) environment for 1 year, and can be stored at 2-8 ℃ for longer periods of time. When the solution is stored at low temperature, it should be left at room temperature for a period of time before use, and then restored to room temperature before use.2. This reagent kit can selectively recover all DNA fragments from the solution. If you need to selectively recover specific fragments while removing other fragments of different sizes, please choose our company's gel recovery reagent kit.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 the Buffer PB. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. The recovery efficiency is related to the initial amount of DNA and the elution volume. The smaller the initial amount, the smaller the elution volume, and the lower the recovery rate.6. All centrifugation steps can be performed at room temperature.Operation steps:1. Estimate the volume of DNA reaction solution, add 5 times the volume of Buffer PB, and mix thoroughly (without removing paraffin or mineral oil).Note: 1) If the DNA reaction system is 50 µ l (excluding paraffin oil volume), add 250 µ l Buffer PB.2) After adding Buffer PB, check the pH value of the solution. If the pH value is greater than 7.5, add 10-30 to it µ 3 M sodium acetate (pH 5.0) was used to adjust the pH value to 5-7.2. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ Centrifuge at 13000 rpm (~16200 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.3. Add the solution obtained in step 1 to the adsorption column that has been loaded into the collection tube, let it stand at room temperature for 1 minute, centrifuge at 13000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Attention: The volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ l, it can be added in batches.4. Add 500 µ l of Buffer PW to the adsorption column (please check if anhydrous ethanol has been added before use), centrifuge at 13000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column in the recovery tube.Note: If purified DNA is used for salt sensitivity experiments (such as flat end ligation experiments or direct sequencing), it is recommended to add Buffer PW and let it stand for 2-5 minutes before centrifugation.5.13000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.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.). To ensure that downstream experiments are not affected by residual ethanol, it is recommended to open the cover of the adsorption column and place it at room temperature for a few minutes to thoroughly dry the residual ethanol in the adsorbent material at the bottom.6. Place the adsorption column into a new centrifuge tube (provided by oneself), add 30-50 µ l Buffer EB to the middle position of the adsorption membrane by hanging droplets, and let it stand at room temperature for 1 minute. Centrifuge at 13000 rpm for 1 minute and collect DNA solution- Store DNA at 20 ℃.Attention:1) The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (the pH value of water can be adjusted to this range using NaOH).2) To improve the recovery of DNA, the solution obtained by centrifugation can be added back to the adsorption column, left at room temperature for 2 minutes, and centrifuged at 13000 rpm for 1 minute.3) The elution volume should not be less than 30 µ l. A small volume will affect the recovery efficiency... Read More | 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 |