| Description | Protein Labeling Kit (Aggregation-induced emission, AIE) is developed based on the principle of aggregation-induced emission (AIE) and is used for labeling general proteins. Its labeling principle is that the AIE dye in the kit carries active groups, which can react with the primary amino groups (Protein Labeling Kit (Aggregation-induced emission, AIE) is developed based on the principle of aggregation-induced emission (AIE) and is used for labeling general proteins. Its labeling principle is that the AIE dye in the kit carries active groups, which can react with the primary amino groups (lysine residues) on the protein surface to form stable amide bonds. The AIE dye has high reactivity and can quickly label general proteins. The optimal excitation wavelength of the AIE dye is 460 nm, and the emission wavelength range is 570–700 nm, which is suitable for a series of instruments equipped with a blue light excitation wavelength detector. Marking renderings:Notes:After receiving the product, customers should store it according to the temperature indicated on the product label.Customers need to prepare dimethyl sulfoxide (DMSO) reagents and disposable syringe consumables by themselves. Experimental Methods 1.Replacement of protein sample buffer solvent: Before starting to label the protein, ensure that the protein sample buffer is compatible with the kit. If your protein sample buffer contains one or more of primary amino groups (such as Tris and glycine), imidazole, DTT, and β-mercaptoethanol, please replace it with Buffer 1× buffer solution before labeling.2.Preparation of AIE dye stock solution: Add 20 µL of DMSO to the small tube containing AIE dye lyophilized powder, mix well to dissolve, and prepare a 10 mM AIE dye stock solution.Notes: 1) Before dissolving with DMSO, let the AIE dye lyophilized powder return to room temperature and centrifuge for 10 seconds. 2) After dissolution, it can be aliquoted for storage as needed.3.Protein labeling method (recommended) 1) Dilute the protein sample with Buffer 1× to a concentration of approximately 150 µg/mL. 2) Add 10 mM AIE dye stock solution to the above protein sample solution to make its final concentration in the protein sample solution 10 µM (for example, when the volume of the protein sample solution is 1 mL, add 1 µL of 10 mM AIE dye stock solution; the dosage can be adjusted according to the actual situation of the protein sample), and mix well. 3) Incubate at room temperature in the dark for 5–10 minutes. 4) After labeling, filter the labeled solution with a 0.1 µm needle filter to remove excess AIE dye.PrecautionsThe AIE dye is sensitive to humidity, so ensure that it is stored at -20°C in a dry and dark place. Before preparing the stock solution, let the dye return to room temperature and centrifuge for 10 seconds. After preparing the stock solution, aliquot it for storage and use it as soon as possible.The buffer solution (pH = 8.5) provided in the kit is the first choice for the AIE dye to work. You can choose any buffer solution that does not contain primary amino groups, imidazole, DTT, or β-mercaptoethanol as a substitute, but it may affect the labeling efficiency.It is recommended that the minimum concentration of the protein during labeling is not less than 15 µg/mL.Due to the differences in the number and reactivity of amino groups on the protein surface, the labeling effects of different proteins may vary. If the labeling effect is not good, the dye labeling concentration can be appropriately increased or the reaction time can be prolonged.For your safety and health, please wear a lab coat and disposable gloves during operation.This product is only for scientific research purposes... Read More | 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 | Product introduction：PMA qPCR live bacteria detection kit provides an effective means to detect bacterial activity. This kit provides a mixture of PMA dye and SYBR green dye based qPCR. The optimal amount of dye and the number of samples that can be processed may vary depending on the type ofProduct introduction：PMA qPCR live bacteria detection kit provides an effective means to detect bacterial activity. This kit provides a mixture of PMA dye and SYBR green dye based qPCR. The optimal amount of dye and the number of samples that can be processed may vary depending on the type of sample. PMA is a DNA binding dye with high affinity, especially with double stranded DNA. The dye itself has weak fluorescence, but it can emit brighter fluorescence after binding with nucleic acids. PMA is impermeable to the cell membrane, so it can selectively modify the DNA of dead cells with damaged membrane. After bllight (~464 nm) photolysis of PMA modified DNA, the photoreactive azido group on PMA is converted into highly reactive azene radical, which reacts with any hydrocarbon moiety near the DNA binding site to form a stable covalent nitrogen carbon bond, resulting in permanent DNA modification. This modification process will make the DNA insoluble, and it will be lost together with cell debris in the later genomic DNA extraction process. The unbound PMA remaining in the solution reacts with water molecules under strong light irradiation and decomposes into hydroxylamine compounds without cross-linking activity, so that it can no longer covalently bind DNA. Based on this characteristic of PMA, our company combines PMA and qPCR technology to form a new detection method - PMA qPCR, which is used for the screening of live bacteria. At present, the method has been validated in a variety of bacterial strains as well as yeast, fungi, viruses and parasites. The treatment of complex samples, such as feces or soil, may require optimization of sample dilution, dye concentration, and light treatment time. Treatment of diluted samples, such as water testing, may require filtration or concentration prior to dye treatment. Component： Instruction： Precautions before use：1.This live bacteria detection kit distinguishes dead bacteria and live bacteria according to cell membrane permeability. Many methods of killing bacteria cause damage to the cell membrane and are therefore compatible with this kit. But some methods, such as ultraviolet irradiation, may not immediately cause cell membrane rupture. Therefore, before selecting this kit, it is necessary to carry out literature search and pre-experiment to determine whether the kit is suitable for the bacterial type and killing method you choose. 2.After PMA treatment, the bacteria need to be photolyzed to covalently bind the dye to dead cell DNA. Photolysis operations can use blue or white light sources. Generally speaking, the brighter the lamp, the higher the efficiency of the photolysis step. Non-LED lamps ( such as halogen lamps ) may heat your sample and have a negative impact on the analysis. Ice is required to cool the sample during irradiation. 3.Sample can be cryopreservation after photolysis. Frozen samples before PMA treatment photolysis may damage the cell membrane and produce false negative results. If the sample needs to be frozen before detection, it is recommended to perform a pre-experiment first. 4.Part of the mechanism of PMA is to remove PMA covalently modified DNA from the sample by precipitation ; therefore, when extracting genomic DNA, it is necessary to use the same volume of genomic DNA eluent for volume normalization. The positive control can use the genomic DNA of living cells. 5.In order to verify the effectiveness of PMA in the test sample, the Ct ( dCt ) changes between- / + PMA can be compared. Experimental materials ( self-provided )：①Light source ( used for the photolysis step after PMA modified DNA ) ;② Bacterial genomic DNA extraction kit ; Experimental procedure： 1.Suck 10 µL of E.coli bacterial solution in liquid LB medium, and culture E.coli in the bacterial incubator overnight or longer to the logarithmic growth phase ( OD600 ≈ 1.0 ) ; Note : The culture time is adjusted according to the experiment. 2.Two portions of live E.coli, 400 µL each, were placed in a clean centrifuge tube ; 3. ( Recommended ) Preparation of dead E.coli. If the dead E.coli is needed as a control, the dead E.coli can be obtained by heating the living E.coli in a water bath at 95 °C for 5 min, or at 58 °C for 3 h. the subsequent operation of the dead E. coli is the same as that of the living E. coli ; 4.Two copies of live E.coli, one without PMA treatment, and one with 25 µM PMA treatment ( the optimal PMA concentration for treating different types or different sources of bacteria needs to be consulted in the relevant literature ) ; 5.The PMA-treated samples were placed on a shaker at room temperature and incubated in the dark for 10 min to fully mix the dye with the sample ; 6.Exposure of the sample, you can use blue or white light source, irradiation time to explore their own. For example, a 60 W blue light can be used for 15 min. Note : 1 If a halogen lamp is used, we recommend that the PMA-treated sample tube be placed on an ice block 20 cm away from the light source. Ice should be placed in a transparent tray. Adjust the light source to point directly to the sample, photolysis for 5-15 min ; if the bacteria obtained from the environment are directly used for experiments, due to the complexity or turbidity of the environmental samples, the photolysis time needs to be prolonged appropriately. 7.Treated and untreated live E.coli 5000 × g, centrifuged for 10 min, remove the supernatant ; 8.Select the appropriate genomic DNA extraction kit according to the sample type, and use the same elution volume for each group of samples when elution DNA. Note : DNA extraction steps refer to the instructions of the kit used. Part of the mechanism of action of PMA is to remove PMA-bound DNA from the sample by precipitation ; therefore, when extracting genomic DNA, each group should use the same volume of genomic DNA eluent for volume normalization ( the amount of genomic DNA extracted from dead bacteria and live bacteria is inconsistent, so the concentration of the two is significantly different ). 9.Preparation of reaction mixture according to the following system : Note : 1 For the DNA extracted by commercial DNA extraction kit, the qPCR template was optimized with 2 µL as the initial volume ; 2 The template volume should not exceed 10 % of the final reaction volume ; 3 Template concentration : gDNA as template, usually 1-10 ng ; the final concentration of PCR primers is usually 0.4µM, which can get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1µM. 10.Slightly vortex the reaction mixture, transfer the fixed volume to the PCR tube. 11. Test procedureNote : 1 The extension time is adjusted according to the instrument ; the Taq enzyme in mix can be activated within 2 min, but the genomic DNA may require longer denaturation time, which can be increased at this time, and the specific denaturation time can be adjusted according to the sample type. 12. ( Optional ) Data analysis Using live bacteria and dead bacteria as controls, the number of live cells in the sample was analyzed and calculated. It is recommended to verify the suitability of primers and PCR procedures before starting PMA qPCR detection of live bacteria. Calculation of dead and living bacteria control dCt ( 1 ) After the end of qPCR, the Ct value of each sample was calculated by instrument software ; ( 2 ) By calculating the dCt of each control bacteria, it was judged whether PMA successfully inhibited the amplification of dead bacterial DNA. The calculation is as follows : dCt live = Ct ( live, PMA treated ) -Ct ( live, PMA untreated ) dCt die = Ct ( die, PMA treated ) -Ct ( die, PMA untreated ) ( 3 ) The dCt expectation of living bacteria is close to 0 ± 1, which indicates that PMA does not affect the amplification of living cell DNA ; ( 4 ) The expected value of dCt of dead bacteria is greater than 4 ( dCt is 4 means that it is reduced by about 16 times, that is, 94 % of dead bacterial DNA is removed ; a dCt of 8 indicated a decrease of about 250 times, that is, 99.6 % of the dead bacterial DNA was removed ).( 5 ) The dCt of dead bacteria depends on many factors, including : strain / cell type ; the way bacteria are killed ; the concentration of PMA used ; amplified sequence length. 3. Calculation of the proportion of viable ( optional ) bacteria If the control results of dead and live bacteria are normal, the proportion of live bacteria in the sample can be calculated.( 1 ) Calculate the dCt value of the sample : dCt sample = Ct ( sample, PMA treated ) -Ct ( sample, PMA untreated ) ( 2 ) Conversion of dCt value to live bacteria ratio : PMA inhibition multiple = 2 ( sample dCt ) Viable bacteria % = 100 / PMA inhibition multiple 14. ( Optional ) Calculate the absolute number of live bacteria If you want to calculate the absolute number of viable bacteria in the sample, you need to use a known number of target bacteria genomic DNA to make a standard curve. It is recommended that the diluted concentrations of several groups of genomes are within the range of the qPCR analysis system. ( 1 ) qPCR was performed with the appropriate genome, and the Ct value was used as the ordinate, and the number of cells was used as the abscissa. The R2 value is calculated to determine the linearity, and the slope and y-axis intercept are displayed.( 2 ) Calculate the copy number of the experimental samples : Ct = slope * cell number + y axis intercept ( y = mx + b ) Bacterial count sample = ( Ct-y axis intercept ) / slope Note : The live bacterial DNA was not lost during the purification process. Examples : Product parameters：Pma: ex = 464 nm; Ex/em = 510/610 nm (following photolysis and reaction with dna/rna)Scope of application：Live bacteria detection Matters needing attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 2.the kit components contain fluorescent dyes, and attention should be paid to avoiding light during use and preservation ; 3.For your safety and health, please wear experimental clothes and disposable gloves... Read More | This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization step, which can significantly reduce the background and enhance the brightness of the target band. P665901Component20 TStorageP665901ASilver Stain Sensitizer (500×)2×1 mLRTP665901BSilver Stain Enhancer3 mLRTP665901CSilver Stain2×250 mLRTP665901DSilver Stain Developer4×125 mLRT Matters needing attention1. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% acetic acid) in advance.2. Please use deionized water and clean glass or plastic containers during operation, and wear disposable gloves for operation.The entire silver dyeing process needs to be carried out on a shaker, with a rotation speed of about 60 rpm.4. Self prepared ethanol and glacial acetic acid are required.Instructions for useThe dosage of each solution in the following operation steps takes the gel with a size of 8.5 × 5.5 cm and a thickness of 1.0 mm as an example. The gel is immersed in the solution completely, and is operated on a shaker, with a general dosage of 25 ml. For large gel, the dosage of each solution should be scaled up according to the gel volume. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: glacial acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% glacial acetic acid) in advance.1. Water washing: After electrophoresis is completed, wash the gel twice with ultrapure water for 5 minutes each time.2. Fixation: Fix the gel twice with 25 ml of fixative solution for 15 minutes each time.3. Elution: Wash the adhesive twice with eluent, each time for 5 minutes.4. Water washing: Wash the glue twice with ultrapure water, each time for 5 minutes.5. Sensitization: put the gel washed in the previous step into the silver dye sensitization working solution, incubate it accurately for 1 minute at room temperature, and then wash it with ultrapure water for three times, each time for 20 seconds. Preparation of silver staining sensitization working solution: Take 50 µ l Silver Stain Sensitivity (500 x) and add it to 25 ml of ultrapure water, mix well.6. Silver staining: discard ultrapure water and incubate gel in silver staining working solution for 30 minutes. Preparation of silver staining working solution: Take 25ml Silver Stain and add 50 µ l Silver Stain Enhanced to mix well.7. Water washing: Quickly wash the glue twice with ultrapure water, with each washing accurately controlled for 20 seconds.8. Development: Immerse the washed gel in the developer immediately and incubate it at room temperature for 2-3 minutes until the protein strip is clear. Preparation of developer: Take 25ml Silver Stain Developer and add 30 µ l Silver Stain Enhanced to mix well. Attention: Within 30 seconds of development, protein bands begin to appear and continue to develop for 2-3 minutes. If the protein band appears lighter, the development time can be appropriately extended to 5 minutes or more.9. Termination: After washing the developer on the gel with the termination solution, soak the gel in a new termination solution to react for 10 minutes.Experimental imagesSilver staining results of BSA protein samples after 10% SDS-PAGE gel electrophoresisThe molecular weight of BSA protein is about 66 kD, and the loading amounts from left to right are 50 ng, 10 ng, and 5 ng, respectively... Read More | Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to Products contentProducts IntroductionThis kit is suitable for simple, rapid and efficient isolation and purification of DNA/RNA from whole blood, tissue homogenates, swabs, serum, plasma and other cell-free body fluids, etc. The unique buffer system enables the viral nucleic acids in the lysate to bind to the silica gel centrifugal adsorbent columns in a highly efficient manner, and the viral nucleic acids obtained are of high purity and stable quality, free of protein, nuclease and other impurities, and can be used in a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments. It can be used for a variety of routine operations, including PCR, fluorescence quantitative PCR and other experiments.Bring your own instrumentsThermostatic mixer.Pre-experiment Preparation and Important Notes1. Read these instructions carefully before experimenting.2. If Proteinase K is to be stored for a long period of time, please keep it at -20℃.3. Check Buffer RLC for crystallization or precipitation prior to use, and if crystallization or precipitation occurs, redissolve Buffer RLC in a 56°C water bath.4. Pre-treatment of tissue samples: Take 20 mg of tissue samples into 1.5 mL centrifuge tubes (self-provided), add 500 µL of Buffer RLC, and after the tissue homogenizer breaks up, centrifuge the samples for 1 minute at 12,000 rpm (~13,400×g), and take 200 µL of supernatant as samples. procedure1. Take a 1.5 mL centrifuge tube (provided), add 500 µL of Buffer RLC, 200 µL of sample, 20 µL of Proteinase K, vortex for 5 s, and then place it in a thermostatic mixer at 1200 rpm for 10 min at room temperature. Note: For wet swab samples, 200 µL of sample was taken after sufficiently shaking and mixing. Note: For wet swabs, 200 µL was taken from the sample after it was soaked in 400 µL of saline, shaken and mixed thoroughly for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute, and 200 µL was taken for extraction.2. Instantly remove the centrifuge tube and add the solution from step 1 to the Spin Columns DM in the collection tube. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.3. Add 500 µL of Buffer PGWT to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.4. Add 500 µL of Buffer GWT2 to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.5. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the adsorption column at room temperature for 2 minutes and allow to dry.6. Place the column in a new collection tube (RNase-Free Centrifuge Tube), add 40-100 µL of RNase-Free Water to the center of the column membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the nucleic acid solution. Store at -80℃ for a long time... Read More |