| Description | Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances luminescent signal intensity, effectively improving detection sensitivity. It can be used to detect firefly luciferase reporter gene expression in ADCC Reporter Bioassays or cells. G1375755Component10 mL100 mLStorageG1375755AFirefly Luciferase Reaction Buffer (Glow)10 mL100 mL-20℃. Store in the dark.G1375755BFirefly Luciferase Substrate (Glow)1 vial1 vial-20℃. Store in the dark.he main features and differences among three One-Step Firefly Luciferase Reporter Gene Assay Kits from Aladdin are as follows:For high luminescent signal requirements and detection within 30 minutes: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760)For both luminescent signal and stability requirements, suitable for batch or continuous operation: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Cat. No. F773892)For standard luminescent signal requirements: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow) (Cat. No. G1375755)For routine laboratory detection, the Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760) is the recommended priority. Product Cat. No.Product NameLuminescence TypeSignal IntensitySensitivityHalf-LifePackagingSuitable for Batch SamplesOptimal Use After Mixing & Storing at -20°CF773892Firefly Luciferase Reporter Gene Assay KitGlowRelatively HighRelatively SensitiveUp to 2h1 bottle Buffer1 vial SubstrateVery Suitable1 MonthG1375755Firefly Luciferase Reporter Gene Assay Kit (Glow)GlowHighSensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekB1375760Firefly Luciferase Reporter Gene Assay Kit (Glow Bright)GlowVery HighHighly Sensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekInstructions for Use:1. User-Supplied MaterialsPBSMultichannel pipetteWhite or black opaque cell culture microplateLuminometer or microplate reader equipped with a luminescence detection module.2. Preparation Before Assay(1) Initial Reconstitution: Upon first use, transfer the entire contents of the Firefly Luciferase Reaction Buffer (Glow) bottle into the Firefly Luciferase Substrate (Glow) vial. Mix thoroughly until the substrate is completely dissolved. Aliquot the reconstituted detection reagent as needed. For long-term storage, aliquot and store at -70°C. For short-term storage, store aliquots at -20°C for no longer than one month. Use reconstituted reagent promptly.(2) Pre-assay: Before each experiment, equilibrate the aliquoted, frozen detection reagent to room temperature.3. Procedure(1) Equilibrate Plates: Remove the cell culture plate from the incubator and allow it to stand at room temperature for 5-15 minutes.Note: Use white or black opaque cell culture plates to minimize signal interference between wells.(2) Add Detection Reagent: Using a multichannel pipette, add the equilibrated Firefly Luciferase Reaction Buffer (Glow) containing the substrate to each cell culture well. The volume added should be equal to the volume of the culture medium in the well. For example:* 96-well plate: Typically add 80-100 µL culture medium; add 80-100 µL detection reagent.* 384-well plate: Typically add 20-30 µL culture medium; add 20-30 µL detection reagent.(3) Mix to Lyse Cells: To ensure complete cell lysis, place the plate on an orbital shaker or an instrument with shaking capability. Shake at medium to high speed at room temperature for 5 minutes.Note: A shaking time of 5 minutes is recommended. This can be adjusted based on cell density to ensure complete lysis and stable luminescent results.(4) Detection: Following the shaking step, immediately measure firefly luciferase reporter gene activity using a luminometer or microplate reader.Note: For optimal results, perform the detection as soon as possible after adding the detection reagent.PrecautionsInstrument Selection: Any instrument capable of detecting chemiluminescence is suitable for use with this kit. However, background signal levels and measured values for identical samples may vary between different detectors. Values obtained from different instruments for the same sample are not directly comparable. To prevent well-to-well interference, the use of opaque white or black cell culture plates is strongly recommended.Consistent Conditions: Luminescent signals can be influenced by environmental factors such as medium components and temperature. Ensure consistent detection conditions across all samples within an experiment.Temperature Sensitivity: The enzymatic reaction is temperature-sensitive. It is essential to equilibrate both the detection reagent and the cell culture plate to room temperature before adding the reagent and performing the assay.Reagent Stability: To maintain the stability of the luciferase assay reagent, aliquot it appropriately after reconstitution. For long-term storage, store aliquots at -70°C protected from light. For short-term storage, store at -20°C for no longer than one month. Use aliquots promptly and avoid repeated freeze-thaw cycles.Multiple Plate Assay: If detecting multiple cell culture plates simultaneously, strive to maintain consistent incubation time after adding the detection reagent to each plate before reading. This ensures optimal and comparable results across plates.Emission Wavelength: The bioluminescence catalyzed by firefly luciferase exhibits a peak emission wavelength of 560 nm... Read More | When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in When apoptosis occurs, some DNA endonucleases will be activated. These endonucleases will cut off genomic DNA between nucleosomes and produce 180 bp-200 BP DNA fragments, which appear as a specific ladder pattern in agarose gel electrophoresis. When double strand or single strand breaks occur in genomic DNA, a large number of sticky 3'-oh ends will be generated, which can interact with YF under the catalysis of deoxyribonucleotide terminal transferase (TDT) ®/ CY dUTP binding can directly detect apoptotic cells by fluorescence microscopy or flow cytometry. This kind of method is called terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Because normal or proliferating cells have almost no DNA breaks, there is no 3'-oh formation and they can rarely be stained. TUNEL method can stain intact single apoptotic nuclei or apoptotic bodies in situ, can accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small number of apoptotic cells, so it is widely used in the study of apoptosis. This kit has a wide range of applications and can be used to detect apoptosis in frozen or paraffin sections, as well as cultured adherent cells or suspended cells. It can selectively detect apoptotic cells, but not necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment. This kit detects cell apoptosis with a short time-consuming, one-step staining reaction and can be detected after washing. Component： Instruction： Experimental materials (self provided)PBS buffer (1 x, pH~7.4). 0.2% Triton X -100 (PBS formulation). 0.1% Triton X -100 (PBS formulation, containing 5 mg/mLBSA)4% paraformaldehyde (prepared with PBS)Immunohistochemical penDewaxing solvent (paraffin section sample)Related reagents for paraffin section processingAnti fluorescence quenching and sealing agent. ddH2Oexperimental design. A. Positive control:Prepare positive control slides using DNaseI treatment. DNaseI can digest single or double stranded DNA and expose the 3 '- OH end, artificially causing cell apoptosis. One experiment per time is sufficient. (To verify if there are any issues with the experimental operation and reagent kit)B. Negative control:Use TUNEL Reaction Buffer without TdT Enzyme and replace TdT Enzyme with ddH2O. (Mainly to exclude non-specific staining caused by cell apoptosis, operational processes, and other reasons; and to adjust the exposure intensity of the shooting.)C. Experimental processing group.The experimental group operated normally according to the instructions.D. Experimental control group.The experimental group operated normally according to the instructions.Experimental steps1. Sample preparation:(1) For adherent cells or cell smearsa. Clean once with PBS.Note: If you are concerned that the cells on the cell smear may not adhere firmly, you can dry the sample to make the cells adhere more firmly.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at 4 ℃ for 30 minutes. Clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Clean twice with PBS.d. Step 2: TUNEL reaction.(2) For suspended cells or cell suspensionsa. Collect cells (3-5 x 106 cells), centrifuge at 1000 rpm for 5 minutes, and wash twice with PBS.b. Fixation: Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and resuspend the cells thoroughly. Fix at 4 ℃ for 30 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.c. Translucency: Add an appropriate amount of 0.2% Triton X -100 (prepared with PBS) and let it penetrate at room temperature for 20 minutes. Centrifuge at 2000 rpm for 5 minutes and clean twice with PBS.d. Step 2: TUNEL reaction.(3) Paraffin tissue sectioninga. Dewaxing and hydration: Place the sliced samples sequentially in xylene I (10 min) → xylene II (10 min) → 100% ethanol I (5 min) → 100% ethanol II (5 min) → 95% ethanol (5 min) → 90% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min) → ddH2O rinse for 5 min, rinse twice.Note: Xylene is toxic and volatile. Please perform this operation in a fume hood.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(4) Frozen tissue sectionsa. Fixation: Take out frozen sections and warm them back to room temperature. Add an appropriate amount of 4% paraformaldehyde (prepared with PBS) and fix at room temperature for 30 minutes. Wash twice with PBS for 10 minutes each time.Note: If you are concerned that formaldehyde cleaning may not be clean enough, it may affect the final dyeing effect. After formaldehyde fixation is completed, an appropriate amount of 2 mg/mL glycine can be added and washed for 10 minutes to neutralize the residual fixing solution, and then PBS cleaning can be carried out.b. Use filter paper to dry the liquid around the sliced sample, and circle the sample contour with an immunohistochemical pen for downstream transparency and labeling.Note: If it is found that the contour circle of immunohistochemistry strokes is damaged in subsequent experimental operations, it needs to be redrawn in a timely manner.c. Transparency: Dilute 2 mg/mL of ProteinaseK solution with PBS in a ratio of 1:100 to a final concentration of 20 µ g/mL. Add 100 µ L dropwise to each sample to cover all sample areas. Incubate at 20-37 ℃ for 20 minutes.Note: Protein K can penetrate the cell membrane and nuclear membrane, allowing subsequent staining reagents to fully enter the nucleus for reaction and improve labeling efficiency. An excessively long incubation time increases the risk of tissue slices falling off the carrier film during subsequent washing steps, while a too short incubation time may result in insufficient permeability treatment and affect labeling efficiency. To obtain better results, the concentration, incubation time, and temperature of Protein K need to be optimized according to different types of tissue samples.d. Wash the slices twice with PBS, each time for 5 minutes. Use filter paper to remove excess liquid, and place the processed sample in a wet box to keep it moist.Note: Protein K must be washed thoroughly in this step, otherwise it will seriously interfere with subsequent labeling reactions.e. Step 2: TUNEL reaction.(5) Positive treatment (only the positive control is subjected to this step, and other samples are directly subjected to the TUNEL reaction step)a. Dilute 10 x DNase I Buffer with ddH2O in a ratio of 1:10 to 1 x DNase I Buffer for later use.b. Drip 100 µ L of 1xDNase I Buffer onto the processed sample, covering all sample areas, and equilibrate at room temperature for 5 minutes.c. Dilute DNase I (2 U) with 1 x DNase I Buffer at a ratio of 1:100/ µ L) A working solution with a final concentration of 20 U/mL.d. Discard the buffer and add 100 µ Incubate DNase I working solution with a concentration of 20 U/mL at room temperature for 10 minutes.e. Discard DNase I working solution and clean twice with PBS.f. Step 2: TUNEL reaction.2. TUNEL reaction(1) Prepare TUNEL reaction solution (ready to use): / 1 sample 5 sample 10 sample TdT enzyme 1 µL 5 µL 10 µL YF®488/555/594/640 TUNEL Reaction Buffer 49 µL 245 µL 490 µL TUNEL Total volume of reaction solution 50 µL 250 µL 500 µL (2) For adherent cells, cell smears, or tissue sectionsa. Add 50 to each sample µ L TUNEL reaction solution, evenly cover the sample with the reaction solution. The appropriate time for dark incubation at 37 ℃ (recommended staining time for cells is 30 minutes to 1 hour, and tissue staining time is 2 hours).Note: 50 µ L TUNEL reaction solution is suitable for smear, slicing, or 96 well plates (other different well plates can adjust the volume of TUNEL reaction solution appropriately to cover cells). If the sample to be tested is a smear, slice, or in a 24 well plate, 12 well plate, or 6 well plate, anti evaporation film can be used, or self sealing bags or other appropriate materials can be used to cut circular plastic sheets slightly smaller than the holes. After adding TUNEL reaction solution dropwise, cover the sample to prevent the evaporation of TUNEL reaction solution and make the TUNEL reaction solution evenly cover the sample.b. Discard the TUNEL reaction solution, wash twice with PBS, and then wash three times with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mL BSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. (Optional) Add an appropriate concentration of 5 to each sample µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes. After staining, discard DAPI staining solution and wash twice with PBS for 5 minutes each time.d. (Optional) Slice sealing: Add 50 drops to each sample µ L anti fluorescence quenching sealing agent (anti fluorescence quenching sealing agent may not be suitable for certain dyes, it is recommended to conduct pre experimental testing for compatibility before the experiment), cover the cover glass, gently tap the cover glass with the blunt end of tweezers to remove bubbles and ensure complete sealing.e. Use filter paper to remove excess liquid and add 100 to the sample area µ Keep the sample moist with PBS and immediately observe under a fluorescence microscope.(3) For suspended cells or cell suspensionsa. Add 50 to each sample tube µ Gently resuspend cells in LTUNEL reaction solution and incubate at 37 ℃ in the dark for 30-1 hour. Gently resuspend cells with a micropipette every 15 minutes.b. Centrifuge at 2000 rpm for 5 minutes, discard TUNEL reaction solution, and wash twice with 0.1% Triton X -100 (PBS preparation, containing 5 mg/mLBSA) for 5 minutes each time. This way, free unreacted markers can be removed cleanly.c. Add 100 to each sample tube µ L concentration is 5 µ DAPI staining solution with a concentration of g/mL, incubated at room temperature in dark for 5 minutes.d. Join 400 µ L PBS resuspended cells and immediately detected with a flow cytometer or observed under a fluorescence microscope after smearing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. when the staining background is heavy or non-specific staining is obvious, the staining time can be appropriately reduced. 3. it is recommended to add negative control and positive control groups during the experiment. 4. please wear mask and gloves when using component A. if it contacts the skin, please wash it with plenty of water immediately. 5. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 6. for your safety and health, please wear experimental clothes and disposable gloves. Product parameters：590/617nm； Scope of application：Late apoptosis detection, TUNEL Kit... Read More | DescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatableDescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity (sold seperately)0-30 mA variable LED outputNon-magnetic LED baseChemically resistant LED coverPTFE coated cablingPhoto Kitalysis Starter Kitrequired for operation (sold separately). Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Storage Buffer1.25 mLRTR669871HBuffer RW140 mLRTR669871IBuffer RW2 (concentrate)11 mLRTR669871JRNase-Free Water10 mLRTR669871KSpin Columns RS with Collection Tubes50 setsRTR669871LRNase-Free Centrifuge Tubes (1.5 mL)50 EART Product IntroductionThis kit is suitable for effectively purifying total RNA from formalin fixed and paraffin embedded tissues. Suitable for extracting total RNA with improved purity from paraffin embedded tissues or sections less than 30mg. This kit does not require the use of phenol/chloroform extraction or isopropanol precipitation, and can complete the extraction of multiple samples within one hour. This product uses specially optimized lysis solution and protease K to release RNA from formalin fixed or tissue slice samples without overnight operation; After digestion, the sample is incubated at a higher temperature to remove the inhibitory effect caused by formalin cross-linking, effectively releasing RNA from tissue slices and avoiding endangering RNA integrity; The optimized buffer system allows RNA in the lysis solution to specifically bind to the silica gel adsorption membrane, while other pollutants can flow through the membrane; It can be effectively removed through rinsing steps, and the washed RNA can be directly used for experiments such as RT-PCR, Real Time PCR, and Western blot analysis.Self prepared reagents: anhydrous ethanol (newly opened or dedicated for RNA extraction), 10mM PBS (pH 7.4).Preparation and important precautions before the experiment1. Add 0.625ml Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. After obtaining the sample, it should be fixed in 4% -10% formalin as soon as possible, with a suitable fixation time of 14-24 hours. Excessive time can lead to RNA breakage and affect downstream experiments.4. Ensure that the sample before embedding is thoroughly dehydrated, as residual formalin will inhibit the action of Protein K.5. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.Before use, please check if there is any crystallization or precipitation in Buffer GTL, Buffer GL, and Buffer DS. If there is any crystallization or precipitation, please dissolve Buffer GTL, Buffer GL, and Buffer DS again in a 56 ℃ water bath.Operation steps1. Sample processing1a. Paraffin embedded sample: Use a surgical knife to trim off excess paraffin from the tissue block, expose the tissue, and cut into 5-10 µ m thin slices.Attention: If the surface of the sample has already been exposed to air, please discard 2-3 pieces that come into contact with the air and do not use them.1b. Samples in fixed solutions such as formalin: Take approximately 20mg of the sample, cut it into small pieces, place it in a centrifuge tube, and add 500 µ 10mM PBS (PH7.4), vortex oscillation, centrifugation at 12000 rpm (~13400 × g) for 1 minute, discard the supernatant, repeat 3 times, and proceed directly to step 3.2. Choose option A or option B to remove paraffinOption AA1. Take approximately 1 × 1cm2 of slices (4-5 slices in total) and place them in a centrifuge tube (prepared by oneself), then add 500 slices µ L Buffer DS, vortex oscillation for 10 seconds. Incubate at 56 ° C for 3 minutes.Centrifuge at A2.12000 rpm for 2 minutes, be careful to discard the supernatant and avoid attracting sediment.Option BB1. Take approximately 4-5 slices of approximately 1 × 1 cm2 and place them in a centrifuge tube (self prepared). Add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds.B2.Centrifuge at 12000 rpm for 2 minutes, be careful to remove the supernatant and avoid removing sediment.B3. Add 1ml of anhydrous ethanol, vortex and shake well. Centrifuge at 12000 rpm for 2 minutes, discard the supernatant, and be careful not to absorb or discard the sediment.B4. Open the tube cover and incubate at room temperature or up to 37 ° C for 10 minutes until there is no ethanol residue.3. Add 150µ L Buffer GTL, resuspended precipitation; Join 10µl Protein K, vortex oscillation mixing.4.Incubate at 56 ℃ for 15 minutes until the sample is completely dissolved. Incubate at 80 ℃ for 15 minutes. Short centrifugation allows the solution on the tube wall to be collected to the bottom of the tube.Note: 1) The purpose of this step is to repair nucleic acids denatured by formaldehyde. Incubating at a high temperature or for too long may cause RNA breakage, resulting in RNA fragments.2) The sample incubated at 56 ℃ can be placed at room temperature until the temperature of the water or dry bath reaches 80 ℃, and then the sample can be incubated at 80 ℃.5. Place on ice for 3 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, be careful not to suck sediment.6. Add 320 to the supernatant µ L Buffer GL, vortex oscillation thoroughly mixed.7. Join 720 µ Mix anhydrous ethanol thoroughly with vortex oscillation.Attention: After adding anhydrous ethanol, there may be a small amount of precipitate precipitation, but it does not affect subsequent operations.8. Add all the solutions obtained in step 7 to the spin columns RS that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. 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.Optional steps: If genomic DNA needs to be removed, the following steps can be followeda. 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.b. Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.c. Add 80 µ l of DNase I mixture directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.d. 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.9. Add 500 to the adsorption column µ Buffer RW2 (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 back into the collection tube.10. Repeat step 9.Centrifuge at 11.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes 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.).12. Place the adsorption column in a new RNase free centrifuge tube, and add 20-50µl to the middle of the adsorption column in the air Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -20 ℃.Note: 1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate. 2) If you want to increase RNA production, you can use 20-50 µ Repeat step 12 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 12... Read More | This kit is used to extract and purify high-quality total RNA from various plants, and is also suitable for the extraction of fungal hyphal RNA. A unique Shredder separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while silica based membrane is This kit is used to extract and purify high-quality total RNA from various plants, and is also suitable for the extraction of fungal hyphal RNA. A unique Shredder separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while silica based membrane is used to adsorb RNA for purification, effectively removing various pollutants such as polysaccharides through washing. The washed RNA can be directly used in various downstream experiments. RNA with a molecular weight greater than 200 bases was extracted using this reagent kit, with high purity and almost no DNA residue. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the remaining DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for experiments such as Northern Blot, Dot Blot, RT-PCR, and in vitro translation. R665489Component50 TStorageR665489ABuffer RL35 mLRTR665489BBuffer RLC35 mLRTR665489CBuffer RW140 mLRTR665489DBuffer RW2 (concentrate)11 mLRTR665489ERNase-Free Water10 mLRTR665489FSpin Columns FL with Collection Tubes50 setsRTR665489GSpin Columns RM with Collection Tubes50 setsRTR665489HRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents:β- Mercaptoethanol, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experiment:To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of RNA extraction.3. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL µ L β Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month. No need to add buffer RLC when using it β- Mercaptoethanol.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. If precipitation occurs in Buffer RL and Buffer RLC, please heat them to dissolve and place them at room temperature.6. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I without RNase.Operation steps:1. Take 50-100 mg of fresh plant tissue, add liquid nitrogen and quickly grind it into powder.2. Collect the ground powder into a centrifuge tube (provided by oneself) and add 600 µ L Buffer RL (check if it is added before use) β- Sulfhydryl ethanol or Buffer RLC, vortex oscillation causes it to fully decompose.Attention:1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for the lysis of most plant tissues. However, in some plant tissues (such as corn endosperm), due to the unique secondary metabolites, guanidine isothiocyanate causes precipitation in the sample, resulting in poor RNA extraction efficiency. In this case, Buffer RLC can be added instead of Buffer RL.2) Incubating at 56 ℃ for 1-3 minutes helps with tissue lysis, but plants with high starch content should not be subjected to high-temperature incubation.3. Transfer all the liquid obtained in step 2 to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (provided by oneself).Attention:1) When aspirating liquid, the tip of the gun can be cut off for easy sampling.2) Spin Columns FL can remove most of the fragments, but there will still be a small amount flowing out. After centrifugation, precipitation will form in the collection tube. When proceeding to the next step, be careful not to absorb the sediment.4. Add 0.5 times the volume of anhydrous ethanol to the clean cracking solution obtained in step 3 and quickly mix well. Attention: Adding ethanol may cause precipitation, but it does not affect subsequent experiments.5. Add all the solutions obtained in step 4 to the spin columns RM that have been loaded into the collection tube. If it is not possible to add all the solutions to the adsorption column at once, please transfer them in two separate steps. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add 700 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube. Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 6 with the following steps.1) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.2) Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1 U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.Attention:The above system is configured according to our company's DNase I reaction system. Please refer to the corresponding instructions for other company products.3) Add 80 µ l of DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.4) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.7. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Repeat step 7.Centrifuge at 9.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly dry the anhydrous ethanol in the column.Attention: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.).10. Place the adsorption column in a new RNase free centrifuge tube, and add 30-50 to the middle of the adsorption column in the air 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 10 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 10... Read More |