| Description | 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 | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat solubility, can easily penetrate cell membranes, and has strong and stable green fluorescence. PKH67-labeled cells can be used for in vitro and in vivo proliferation studies, and have the function of not staining neighboring cells. In the process of cell division and proliferation, the fluorescence intensity of PKH67 will gradually decrease as the cells divide. The labeled fluorescence can be evenly distributed to the two sub-generation cells, so its fluorescence intensity is half that of the parent cell. According to this feature, It can be used to detect cell proliferation, cell cycle estimation and cell division, etc. The fluorescence of PKH67-labeled cells is very uniform, and the fluorescence distribution of sub-generation cells after division is also more uniform. In the process of cell division and proliferation, PKH67-labeled fluorescence can be evenly distributed between the two sub-generation cells, and the fluorescence intensity becomes half of that of the parent cell. According to the difference in fluorescence intensity, the undivided cells can be detected by flow cytometry. One time (1/2 the fluorescence intensity), the second time (1/4 the fluorescence intensity), three times (1/8 the fluorescence intensity), and more divisions of cells. PKH67 can detect splits up to six times or even more. In addition to the detection of cell proliferation, PKH67 can also be used for in vitro tracking of cells. After labeling, the fluorescence expression is stable in the cell, and the positive labeling rate is over 98%. The labeled cells have good morphology, which can effectively observe the cells in vitro. Induce differentiation; or inject labeled cells into the body, it can effectively show the migration and differentiation of transplanted cells in living tissues. PKH67-labeled cells can be used for in vivo observation for as long as several weeks. It is often used for in vivo cell detection experiments and experiments to observe long-term cell activity using fluorescence electron microscope. PKH67 is less toxic and does not affect cell proliferation. This method is simple to operate, does not use radioactive isotopes, and poses no safety hazards. You can get the desired experimental data faster, more accurately and more safely.Due to the longer length of the charcoal tail, internal studies have shown that PKH67 is less transferred between cells than PKH2. In in vivo studies using PKH1 and PKH2, the fluorescence intensity will slowly lose. Since this is a behavioral characteristic of green cell linker dye rather than red cell linker dye, PKH67 will have similar properties. The correlation between the in vitro cell membrane retention of non-dividing cells and the in vivo fluorescence half-life reveals that the in vivo fluorescence half-life of PKH67 is 10-12 days. Other green cell linker dyes with similar half-lives have been used to monitor the transport of lymphocytes and macrophages in the body within one to two months. The results indicate that PKH67 can also be used for medium-term in vivo tracking studies.The dye can stably bind to the lipid region of the cell membrane and emit fluorescence, and is mainly used for cell labeling in vitro, cell proliferation research in vitro, and cell tracing research in vivo and in vitro. The fluorescence half-life of PKH67 in vivo is 10-12 days. Compared with PKH-67, PKH-26 has a longer half-life, and the half-life of PKH26 labeled on rabbit red blood cells is more than 100 days. Especially suitable for in vitro proliferation research and long-term in vivo cell tracking research. After PKH67 labels the cells, flow cytometry is usually used for cell proliferation detection.Kit components0.1ml kits: P266290A-0.1ml P266290B-10ml1ml kits: P266290A-1ml P266290B-60mlDyes with A suffix and diluents with B suffix are used togetherPKH67 labeled cells show green fluorescence, the fluorescence wavelength: λex=490 nm, λem=502 nm.Storage conditions: -20℃ protected from light, valid for 1 yearPrecautions●Staining concentration varies according to the type of cell and the number of cells in each well.● The prepared PKH67 mother liquor is very easy to dissolve. It is recommended to store in aliquots and freeze-dry at ≦-20℃.● PKH67 working solution should be prepared for immediate use, and cannot be prepared in advance, because PKH67 will decompose due to the absorption of water and affect the dyeing effect.● PKH67 is easily decomposed and will deteriorate quickly in the water solution. Please avoid contact with water during use of mother liquor. The working fluid is in contact with the water during the process of labeling the cells within the permitted time range.● PKH67 fluorescent dye is a DMSO solution. It will solidify and stick to the bottom, wall or cap of the tube at a lower temperature such as 4℃ and ice bath. After being taken out of the refrigerator, it will return to room temperature and become After the liquid is in the state, remove the cap from the bottom of the tube. It can be used after it has completely melted in a 37°C water bath.● The number of generations or time that can be traced after different cell types are marked is quite different. Please make a test based on the actual situation or reference documents.Instructions1. Staining solution preparation:(1) Take out the PKH67 reagent from the refrigerator, let it stand for a few minutes to room temperature, or after a 37°C water bath, leave the tube containing PKH67, and be sure to leave the tube for a few minutes before opening the lid to allow the reagent to fully fall into the tube The lid can only be opened after the bottom.(2) According to the number of cell samples to be tested, dilute the probe 10 times with the diluent, and then use a suitable solution (such as non-clear medium, HBSS or PBS) to dilute the PKH67 mother liquor 25 times to prepare a stain Work fluid. The best working solution concentration should be adjusted according to different cells and your own experimental system. Generally, the cells can be diluted 250 times according to the final concentration of the mother liquor in the kit. Some cells may need to increase the concentration appropriately.2. Cell staining(1) Resuspend the prepared cells to be tested in 100µl of staining solution to a cell concentration of about 107/ml. You can also perform in-situ staining, as long as the staining solution is enough to cover the cells.(2) Culture the cells at 2～8℃ for 15～30 minutes. The best culture time is different for different cells.It is recommended to incubate the labeled cells in the staining solution at 37°C for 5 minutes, and then at 4°C for 15 minutes.Low-temperature incubation can reduce the endocytosis of the dye by the cells, help the dye to label the plasma membrane, and reduce the possibility of the dye localizing to cytoplasmic vesicles.(3) After separation, remove the supernatant, collect the cells, wash the cells 1-2 times with PBS or non-clear medium, and finally add PBS or non-clear medium to resuspend the cells.(4) Take 500µl of cell suspension and test with flow cytometer. Ex/Em=490/502nm.(5) Subsequently, the cells can be cultured according to the normal culture method.(6) The labeling effect can be directly observed under a fluorescence microscope, or the cell proliferation can be detected by a flow cytometer after an appropriate period of culture, or used for cell fluorescence traces for other specific experimental purposes... Read More | R669988 Component 50T Storage R669988A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. R669988B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. R669988C Buffer RL 35 mL RT R669988D Buffer RLC 35 mL RT R669988E Buffer RW1 40 mL RT R669988F Buffer RW2 (concentrate) 11 mL R669988 Component 50T Storage R669988A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. R669988B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. R669988C Buffer RL 35 mL RT R669988D Buffer RLC 35 mL RT R669988E Buffer RW1 40 mL RT R669988F Buffer RW2 (concentrate) 11 mL RT R669988G RNase-Free Water 10 mL RT R669988H Spin Columns FL with Collection Tubes 50 sets RT R669988I Spin Columns RM with Collection Tubes 50 sets RT R669988J RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProductsThis kit is used for the extraction and purification of high-quality total RNA from a variety of plants, and is also suitable for the extraction of fungal mycelial RNA. The unique separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while the silicon matrix membrane is used to adsorb the RNA for purification, so that various contaminants, such as polysaccharides, are effectively removed by washing, and the eluted RNA can be directly used in various downstream experiments. The molecular weight of RNA extracted by this kit is more than 200 bases, with high purity and almost no DNA residue. For RNA experiments that are very sensitive to trace DNA, the residual DNA can be removed by digestion on a column using RNase-free DNase. The extracted RNA can be used in Northern Blot, Dot Blot, RT-PCR and in vitro translation experiments.Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.2) RNase-free water should be used to prepare the solution.(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.(2) Glassware should be dry-roasted at 180°C for 4 hours before use, and plasticware can be soaked in 0.5M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.3) RNase-free water should be used to prepare the solution.(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.3. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.4. Please add β-mercaptoethanol to Buffer RL before use, add 10µl of β-mercaptoethanol to 1ml of Buffer RL, it can be stored for 1 month at room temperature. Buffer RL with β-mercaptoethanol can be stored at room temperature for 1 month. β-mercaptoethanol is not required for use of Buffer RLC.5. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.6. If precipitation occurs in Buffer RL and Buffer RLC, heat to dissolve and leave at room temperature.7. All centrifugation steps are carried out at room temperature and all steps are performed quickly. Procedure1. 50-100 mg of plant tissue is quickly ground to a powder in liquid nitrogen and added to 600 µl of Buffer RL (check for addition of β-mercaptoethanol before use) or Buffer RLC. vortexing and oscillating to allow for adequate lysis.Note: 1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for lysis of most plant tissues. However, in some plant tissues (e.g. endosperm of corn), due to the special secondary metabolites, guanidine isothiocyanate causes precipitation of the sample, resulting in poor RNA extraction, in this case, Buffer RLC can be added instead of Buffer RL.2) Incubation at 56°C for 1-3 minutes helps tissue lysis, but do not incubate at high temperatures for plants with high starch content.2. Transfer all the liquid obtained in step 1 to an adsorption column (Spin Columns FL) that has been loaded into a collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (supplied).Note: 1) The tip of the tip of the gun can be cut off when aspirating liquids to facilitate sampling.2) Spin Columns FL removes most of the debris, but a small portion will still flow out and a precipitate will form in the collection tube after centrifugation, so be careful to avoid aspirating the precipitate when proceeding to the next step.3. Add 0.5 times the volume of anhydrous ethanol to the clean lysate obtained in step 2 and mix rapidly.Note: Precipitation may occur upon addition of ethanol, but does not affect subsequent tests.4. Transfer the solution obtained in the previous step to the Spin Columns RM in the collection tube. If it is not possible to add all of the solution to the column at one time, centrifuge the column at 12,000 rpm for 15 seconds in two batches, discard the waste solution and put the column back into the collection tube.5. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and make a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid and put the column back into the collection tube.9. Add 500 µl of Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 15 seconds, and discard the waste solution.10. Repeat step 9.11. Place the adsorbent column back into the collection tube, centrifuge at 12,000 rpm for 1 minute, and allow the column to come to room temperature for a few minutes to thoroughly dry out the anhydrous ethanol in the adsorbent column.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column into a new centrifuge tube, add 30-50 µl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... Read More |