| Description | Inquire | D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H Buffer EB 4 mL RT D665729I Buffer PS 10 mL RT D665729J Spin Columns DF 50 Pcs 2-8 ℃ D665729K Collection Tubes 50 Pcs RTProduct Introduction:The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.Self prepared reagents: anhydrous ethanol, 75% ethanol.Preparation and important precautions before the experiment1. Product usage method:(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 µ M-Dissolving Buffer and 300 µ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.Operation stepsThe range of DNA prepared each time is 1 ng-4 µ Between g, the optimal amount is 500 ng-2 µ G.1. Take 20 µ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 µ L.2. Add 2.2 to the DNA sample µ Mix the sample well with the M-Dilution Buffer of l.3.42 ℃ water bath for 30 minutes.4. Add 220 to the sample obtained from the previous step µ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.5. Add 480 to the solution in the previous step µ M - Buffer PA, gently mix upside down.6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube µ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, centrifuge at 12000 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 capacity of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.8. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.9. Add 650 to the adsorption column µ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.10.12000 rpm for 2 minutes, discard the waste liquid, and 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 will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air µ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.12. Collect 20 µ Add 2.2 to DNA µ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.13. Add 500 to the solution µ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).14.12000 rpm for 15 minutes and gently discard the supernatant.15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 µ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments... Read More | DescriptionProvides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with KitAlysis High-Throughput Screening Kits and KitAlysis 24-Well Reaction Block | 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)... Read More | This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked andHomogenized samples, in their unique high salt state, RNA specifically binds to silicon matrix membranes, greatly reducingEffectively removing organic solvent contamination while removing protein contamination, resulting in higher purity and quality of RNA. bookThe product can quickly extract total RNA from various cells or tissues, and can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time,Can handle multiple different samples simultaneously. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the residual DNA can be utilizedUsing DNase without RNase for digestion and removal on the column, the extracted RNA can be directly applied to RT-PCR Experiments such as Northern Blot, Dot Blot, and in vitro translation. U665516 Component 50 T Storage U665516A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. U665516B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. U665516C TRIzon Reagent 60 mL 2-8℃. Protect from light. U665516D TRIzon PaI™ 10 mL 2-8℃. Protect from light. U665516E Buffer RW1 40 mL RT U665516F Buffer RW2 (concentrate) 11 mL RT U665516G RNase-Free Water 10 mL RT U665516H Spin Columns RM with Collection Tubes 50 sets RT U665516I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTPreparation and important precautions before the experiment:1.To prevent RNase pollution, attention should be paid to the following aspects:1) RNase's plastic products and gun heads to avoid cross contamination.2) Prepare the solution using water without RNase.3) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction.3. If TRIzon Reagent is found to have precipitates before use, it can be dissolved in a water bath at 56 ℃ for a few minutes.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Usage：1. Sample processing1a. Organization: 30-50 mg of tissue is thoroughly ground in liquid nitrogen and 1 mL of TRIzon Reagent is added, or 1 mL of TRIzon Reagent is added to the tissue sample and homogenized. Attention: The sample volume should not exceed 10% of the volume of TRIzon Reagent.2a. Single layer cell culture: Remove the culture medium and add an appropriate amount every 10 cm ² Add 1 mL of TRIzon Reagent.3a. Cell suspension: Collect cells by centrifugation. Add 1 mL of TRIzon Reagent to every 5 × 10 µ m cell.2. After adding TRIzon Reagent, repeatedly blow a few times to fully crack the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Add 200 to every 1 mL of TRIzon Reagent µ LTRIzon PaI ™， Cover the tube tightly, vigorously shake for 15 seconds, and let it sit at room temperature for 2 minutes.4. Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer, and the upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Move the upper aqueous phase to a new RNase Free centrifuge tube (provided).5. Add an equal volume of 70% ethanol (prepared without RNase water) to the obtained aqueous solution, invert and mix well.6. Add all the solutions obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Preparation of DNase I mixture: Take 52 µ LRNase Free Water, add 8 to it µ L 10 x Reaction Buffer and 20 µ L DNase I (1 U/ µ L) Mix well and prepare to a final volume of 80 µ The reaction solution of L.9. Directly add 80 µ L DNase I mixture to the adsorption column and incubate at 20-30 ℃ for 15 minutes.10. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.11. Add 500 to the adsorption column µ L Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.12. Repeat step 11.Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes and thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion,. )PCR, etc.14. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ L. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 14 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 14... Read More |