| Description | Inquire | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... 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 | This product is a cDNA first strand synthesis kit specially prepared for the first step experiment of two-step RT-PCR. This product contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including HiFi MMLV reverse transcriptase, reaction buffer, This product is a cDNA first strand synthesis kit specially prepared for the first step experiment of two-step RT-PCR. This product contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including HiFi MMLV reverse transcriptase, reaction buffer, primers, dNTP, etc. The mutated HiFi MMLV reverse transcriptase RNase H activity is deficient, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi MMLV reverse transcriptase has strong thermal stability and can yield high yields of cDNA, making it simple and convenient to use. This system has high compatibility with subsequent PCR and quantitative PCR experiments, and is suitable for various DNA polymerase reactions. H665693 Component 100 T Storage H665693A HiFi-MMLV, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665693B 5×RT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665693C Primer Mix 240 µL -20℃. Avoid freeze/thaw cycle. H665693D dNTP Mix, 2.5 mM Each 500 µL -20℃. Avoid freeze/thaw cycle. H665693E DTT, 0.1 M 240 µL -20℃. Avoid freeze/thaw cycle. H665693F RNase-Free Water 1 mL -20℃. Avoid freeze/thaw cycle. Product features:·RNase H -: Mutated HiFi MMLv reverse transcriptase with reduced RNase H activity, making it easier to obtain full-length cDNA.·Easy to use: The reagent kit contains all the reagents required for reverse transcription, except for RNA templates.Notes:1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.Usage:Attention: 10 ng-5 µ G Total RNA can establish 20 µ Reaction system, if the total RNA content is greater than 5 µ g. Please expand the reaction system proportionallyi Steps for reverse transcription:1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.2. Prepare a reaction system according to the following table, with a total volume of 20 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Primer Mix 2 µl / RNA Template X µl 1 ng-5 µg 5×RT Buffer 4 µl 1× DTT，0.1 M 2 µl 10 mM HiFi-MMLV，200 U/µl 1 µl / RNase-Free Water up to 20 µl / Attention:1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.2) Primer Mix is formulated from Oligo (dT) and Random Primer3. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe. 4. Incubate at 42 ℃ for 30-50 minutes and 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.5. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.ii If the reverse transcription efficiency is low, or the RNA template secondary structure is complex and the GC content is high, the following steps are recommended:1. Dissolve RNA templates, primers, dNTP Mix, DTT, RT Buffer, HiFi MMLV, and RNase Free Water and place on ice for later use.2. Prepare the reaction system according to the following table, with a total volume of 13 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Primer Mix 2 µl / RNA Template X µl 1 ng-5 µg RNase-Free Water up to 13 µl / 3. Incubate at 70 ℃ for 10 minutes and quickly ice bath for 2 minutes.4. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.5. Continue to add the following reagents to the above reaction solution: Reagent 20 µlReaction system Final concentration 5×RT Buffer 4 µl 1× DTT，0.1 M 2 µl 10 mM HiFi-MMLV，200 U/µl 1 µl / Attention:1) If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.2) Primer Mix is formulated from Oligo (dT) and Random primer.6. Gently blow and mix well, incubate at 42 ℃ for 50 minutes, and incubate at 85 ℃ for 5 minutes.7. After the reaction is complete, centrifuge briefly and cool on ice.8. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time... Read More | Lipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Polyunsaturated lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the productionLipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Polyunsaturated lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the production of end products such as malondialdehyde (MDA). Lipid peroxidation may contribute to the pathology of many diseases including atherosclerosis, diabetes, and Alzheimer′s.Lipid peroxidation (MDA) assay kit has been used to determine the levels of malondialdehyde (MDA).Suitability: Suitable for the measurement of malondialdehyde (MDA) in a variety of samples including tissue, cells and plasmaPrinciple: In this kit, lipid peroxidation is determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm)/fluorometric (λex= 532/λem= 553 nm) product, proportional to the MDA present... Read More |