| Description | The commonly used method of eukarYOtic gene expression regulation research is the detection of reporter genes, and bioluminescence is the most commonly used and effective means of reporter gene detection. Luciferase can catalyze the conversion of the substrate luciferin and emit photons. This The commonly used method of eukarYOtic gene expression regulation research is the detection of reporter genes, and bioluminescence is the most commonly used and effective means of reporter gene detection. Luciferase can catalyze the conversion of the substrate luciferin and emit photons. This product provides a rapid, sensitive and stable detection method for the expression of Renilla luciferase reporter gene in mammalian cells. Product characteristic：1.Rapid : Cell lysis was completed within 10-15 min ；2.Convenience : The reagent is easy to prepare, and the sample detection steps are simple；Instruction：1. Cell lysis ( 1 ) Remove the culture medium and gently wash with PBS ( adherent cells can be directly performed this operation, suspension cells should be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products after full pyrolysis were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was moved into a new EP tube for subsequent detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C into renilla luciferase working solution with component B, and the dilution method is to add 1 µL C component to 49 µL B component. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) The cell lysis products were added to the measuring tube according to the volume of 20 ~ 100 µL ( keep the same amount of samples each time ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL renilla luciferase working solution was added to determine the RLU ( Relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). Note : The renilla luciferase working solution cannot be stored for a long time. It is now ready for use and is used once. Component：RenillaLuciferase Lysis Buffer；RenillaLuciferase Assay Buffer；CoelenterazineMatters needing attention：Scope of application： Matters needing attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 2.Due to the influence of temperature on the enzyme reaction, the sample and reagent should be measured after reaching room temperature. 3.The strongest wavelength of bioluminescence catalyzed by renilla luciferase is 480 nm, in order to prevent interference between holes, it is recommended to use white opaque orifice plate ;4. B component is recommended to carry out small batch packing according to the experimental requirements ; 5.It is recommended to use it now to avoid repeated freezing and thawing ; 6.For your safety and health, please wear experimental clothes and wear disposable gloves. Scope of application：Study on gene expression regulation and promoter... Read More | Inquire | B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G Proteinase K 1.25 mL 4×1.25 mL RT B665530H Spin Columns DM with Collection Tubes 50 sets 200 sets RTProduct IntroductionThis reagent kit is suitable for extracting total DNA, including genomic DNA, mitochondrial DNA, and viral DNA, from fresh or frozen whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin), plasma, serum, erythrocyte sedimentation rate brown layer, lymphocytes, cell-free body fluids, and other samples. This product can process 0.1-1 mL of whole blood with a maximum yield of 30% µ g. It can purify DNA with sizes ranging from 100 bp to 50 kb. The purified DNA has high yield and good quality, and can remove protein, pigment, lipid, and other inhibitory impurities to the maximum extent. It can be directly used for PCR, fluorescence quantitative PCR, enzyme digestion, and Southern Blot experiments.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. The sample should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2. This reagent kit can extract up to 0.1-1 mL of whole blood samples or 1 × 107 white blood cells.3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in the Buffer GL. If there is any crystallization or precipitation, please incubate the Buffer GL in a 56 ℃ water bath and dissolve it again.5. The Buffer RCL in the reagent kit cannot be used again after being turbid.Operation steps:1. Sample processing: 1a When extracting 200 uL of blood sample, add the sample to the centrifuge tube (provided) and proceed directly to the next step of the experiment. 1b When the blood sample size is less than 200 µ When L, add Buffer GR to make up for 200 µ L. Proceed to the next step of the experiment. 1c When the blood sample size exceeds 200 µ When L is reached, add 1-2 times the volume of Buffer RCL, gently vortex or invert and mix well. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and carefully discard the supernatant. If there is still red in the sediment, repeat the above steps once. Then add 200 to the precipitate µ Shake the buffer GR until thoroughly mixed before proceeding to the next step of the experiment. 1d If the processed blood sample is anticoagulant from poultry, birds, amphibians, or lower level organisms, its red blood cells are nucleated cells, and the blood sample size is 5-20 µ L. Can be added to Buffer GR to make up to 200 µ Follow up experiments will be conducted afterwards. Note: If downstream experiments are sensitive to RNA, 4 can be added µ L RNase A (100mg/mL) solution, shake for 15 seconds, and leave at room temperature for 5 minutes. RNase A reagent kit is not provided. If needed, you can order it separately from our company, item number: CW0601S.2. Add 20 to the above solution µ L Protein K, mix well.3. Add 200 µ Shake with L Buffer GL until thoroughly mixed. Note: Do not pre mix Protein K and Buffer GL.4.Incubate at 4.56 ℃ for 10 minutes, invert and mix several times during this time. Attention: The DNA production has reached its maximum after 10 minutes of incubation, and further extension of incubation time has no effect on DNA production and purity.5. Add 200 µ L anhydrous ethanol, invert and mix several times. Short centrifugation causes the liquid on the tube wall and wall cover to concentrate at the bottom of the tube.6. Add all the solution obtained in step 5 to the spin columns DM 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.7. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol is 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. Note: If the extracted sample is the blood genome of species such as mice or monkeys that are difficult to remove heme, it is recommended to repeat step 7.8. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol is 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. Note: To further improve DNA purity, repeat step 8.9.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 air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.)10. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) If the final concentration of DNA needs to be increased, the obtained DNA eluent can be added back to the adsorption membrane, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute. 3) Because DNA stored in water is affected by acidic hydrolysis, if long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kitProduct content: M665794Component125 TStorageM665794A2×miRNA qPCR Mixture (ROX)2×750 µL-20℃. Avoid freeze/thaw cycleM665794BReverse Primer, 10 µM60 µL-20℃. Avoid freeze/thaw cycleM665794CddH2O1.5 mL-20℃. Avoid freeze/thaw cycle Product Introduction:This kit uses the principle of SYBR Green I chimeric fluorescent dye method for miRNA fluorescence quantitative PCR detection. The kit includes 2 x miRNA qPCR Mixture and Reverse Primer required for detection. 2 x miRNA qPCR Mixture is a new generation pre mixed form of fluorescence quantitative PCR detection reagent specially developed for miRNA quantitative detection. The fluorescent dye SYBR Green I contained in it can bind to all double stranded DNA, making the product suitable for detecting different target sequences without the need to synthesize specific labeled probes. The GoldStar Taq DNA polymerase is a chemically modified and highly efficient thermal starter enzyme, coupled with a unique buffer system, which enhances reaction specificity, sensitivity, and enables accurate quantification of miRNA over a wider range. The 2x miRNA qPCR Mixture contains ROX dye and is suitable for fluorescence quantitative PCR instruments that require ROX as a calibration dye.Note: This kit must be used in conjunction with the miRNA cDNA first strand synthesis kit.Self prepared experimental materials: qPCR upstream primer.Forward Primer design principles:1. Follow the most common principles of primer design.2.Based on mature miRNA sequences, replacing U with T is the most basic and simplest design method.3.The Tm value of the downstream primer provided in the reagent kit is 63.6 ℃, and the Tm value of the upstream primer should be designed to be around 63.6 ℃ as much as possible.4. If the Tm value of the primer directly designed according to principle "2" is too low, several bases (preferably G or C bases) can be added to the 5 'end of the primer; One or several A bases can also be added at the 3 'end; Alternatively, both the 5 'and 3' ends can be modified simultaneously.5.If the Tm value of a primer designed directly according to principle "2" is too high, several bases can be removed from the 5 'or 3' end of the primer.Notes:1. Before using the reagent, please gently mix it upside down to avoid foaming, and use it after a brief centrifugation.2. The amount of miRNA first strand cDNA added should not exceed 10% of the volume of Real time PCR.3. For special detection systems, high content of cDNA templates can easily lead to non-specific amplification. Dilute cDNA appropriately (10 or 100 times dilution) based on the abundance of detected miRNAs.4. The 2x miRNA qPCR Mixture in this product contains SYBR Green I and ROX dyes. When storing this product or preparing PCR reaction solution, strong light exposure should be avoided.5. Avoid repeated freezing and thawing of this product. Repeated freezing and thawing may cause a decrease in product performance. This product can be stored at -20 ℃ for long-term storage. If frequent use is required in the short term, the 2xmiRNA qPCR Mixture can be stored at 2-8 ℃. However, the Reverse primer still needs to be stored at -20 ℃.Operation steps:1. Melt 2 x miRNA qPCR Mixture and Reverse Primer at room temperature (10 µ M). 2. When using, please gently mix the 2x miRNA qPCR Mixture upside down to avoid foaming, and use after brief centrifugation. If the reagent is not well mixed, its reaction performance will decrease.3. Place the reagent on ice and prepare the reaction system according to the following table: reagent volume final concentration 2×miRNA qPCR Mixture（ROX） 10 µl 1× Forward primer（10 µM） 0.4µl 0.2 µM Reverse primer（10 µM） 0.4µl 0.2 µM MiRNA first strand cDNA X µl — ddH2O up to 20 µl —4. The reaction program is set as follows:Attention!The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes! Note: 1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.2) The annealing temperature should be set at 60-64 ℃ as a reference range. When non-specific reactions occur, the annealing temperature can be increased... Read More |