| Description | Product introduction： The MA qPCR live bacteria detection kit provides an effective means for detecting bacterial activity. The kit provides a mixture of PMA dye and qPCR based on SYBR Green dye. The optimal amount of dye and the number of samples that can be treated may vary depending on theProduct introduction： The MA qPCR live bacteria detection kit provides an effective means for detecting bacterial activity. The kit provides a mixture of PMA dye and qPCR based on SYBR Green dye. The optimal amount of dye and the number of samples that can be treated may vary depending on the type of sample. PMA is a high-affinity DNA-binding dye, especially with double-stranded DNA. The dye itself has weak fluorescence, but it can emit brighter fluorescence after binding to nucleic acids. PMA is impermeable to cell membranes, so it can selectively modify the DNA of dead cells with damaged membranes. After the PMA-modified DNA is photolyzed by blue light ( ~ 464 nm ), the photoreactive azide group on the PMA is converted into a highly reactive nitrene radical, which reacts with any hydrocarbon near the DNA binding site to form a stable covalent nitrogen-carbon bond, resulting in permanent DNA modification. This modification process will make DNA insoluble and lost with cell debris during the later genomic DNA extraction process. The unbound PMA remaining in the solution reacts with water molecules under strong light irradiation to decompose into hydroxylamine compounds without cross-linking activity, so that it can no longer covalently bind to DNA. Based on this feature of PMA, PMA was combined with qPCR technology to form a new detection method, PMA-qPCR, for the screening of live bacteria. At present, the method has been verified in a variety of bacterial strains, yeast, fungi, viruses and parasites. The treatment of complex samples, such as manure or soil, may require optimization of sample dilution, dye concentration, and light treatment time. The treatment of diluted samples, such as water testing, may require filtration or concentration prior to dye treatment. Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the components of the kit contain fluorescent dyes. Avoid light during use and storage. 3. for your safety and health, please wear experimental clothes and disposable gloves.Product parameters:Spectral characteristics :PMA: Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA)Component： PMA：Ex = 464 nm; Ex/Em = 510/610 nm (following photolysis and reaction with DNA/RNA) 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 ( for the photolysis step after PMA modification of DNA ) ; ② Bacterial genomic DNA extraction kit ; ③ effective qPCR primers corresponding to the sample type 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 procedure Note : 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. 13. 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 : Scope of application：Live bacteria detection... Read More | Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and phosphoglucose dehydrogenase. The content of glucose-1-phosphate (1PG/G1P) in the sample can be calculated by detecting the increase in NADPH at 340nm.Composition and preparation of reagent kit: Reagent name Specifications Save requirements Remarks Extraction solution Liquid 100mL x 1 bottle 4 ℃ storage / Reagent 1 Powder mg x 1 tube 4 ℃ storage Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 2 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 3 Liquid 16mL x 1 bottle 4 ℃ storage / Reagent 4 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then add 1 Dissolve 1mL of distilled water for later use. TRC 1 powder 4 ℃ storage Only used to identify whether the reagents in the kit are normal (not involved in result calculation). Usage: Use a pre standard tube (GIP) to shake the powder a few times until it falls to the bottom, then add 0.5mL of distilled water and mix well to dissolveDilute GIP with a concentration of 4mg/mL and then dilute it four times to 1mg/mL for later use: follow the instructions in the sample addition table for the measuring tube operationRequired instruments and supplies:ELISA reader, 96 well plate, desktop centrifuge, adjustable pipette, mortar, ice and distilled water.Determination of glucose-1-phosphate (1PG/G1P) content:1. Sample preparation① Organizational sample:Suggest weighing around 0 1g of tissue, add 1mL of extraction solution, and homogenize in an ice bath. Centrifuge at 12000rpm, 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, it can be extracted in a ratio of tissue mass (g) to extraction solution volume (mL) of 1:5-10.② Bacterial/cellular samples:Collect bacteria or cells into a centrifuge tube first, centrifuge and discard the supernatant; Take about 5 million bacteria or cells and add them to 1mLExtract solution, sonicate bacteria or cells (ice bath, power 200W, sonication for 3s, interval 10s, repeated 30 times); Centrifuge at 12000rpm at 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, extraction can be carried out in a ratio of 500-1000:1 of bacteria/cell quantity (104) to extraction solution (mL).③ Liquid sample: direct detection.2. Machine testing:① Preheat the enzyme-linked immunosorbent assay (ELISA) reader for at least 30 minutes and adjust the wavelength to 340nm.② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table:② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table: Reagent name (µL） Measurement tube Blank tube (only done once) Reagent 1 10 10 Reagent 2 10 10 Reagent 3 150 170 Sample 20 / Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A1 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Reagent 4 10 10 Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A2 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Δ A=(A2-A1) measurement - (A2-A1) blank.[Note] 1 If the difference in Δ A is hovering around zero, the sample size V1 can be increased (such as increasing to 50 µ L, the three phases of the reagent should be reduced while keeping the total volume unchanged), or the sample sampling mass W can be increased. The changed V1 and W need to be substituted into the formula for recalculation.If the A2 value exceeds 1.2, the amount of sample added V1 can be reduced (such as to 10 µ L, the three-phase reagent should be increased while keeping the total volume unchanged), or the sample can be diluted with distilled water (keeping the sample addition system unchanged), and the changed V1 and D need to be substituted into the formula for recalculation.Result calculation:1. Calculated by sample weight:1PG/G1P content (µ g/g fresh weight)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (W × V1 ÷ V) × D=836 × Δ A ÷ W × D2. Calculated by the number of cells:1PG/G1P content (µ g/104 cell)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (500 × V1 ÷ V) × D=1.7 × Δ A × D. 3. Calculated by liquid volume:1PG/G1P content (µ g/mL)=[(Δ A ÷ (ε× d) × V2 × 106 × Mr] ÷ V1=836 × Δ A ε---NADPH Molar extinction coefficient，6.22×103 L/mol/cm； d---96 Orifice plate optical diameter，0.5cm； V---Add volume of extraction solution，1 mL； V1---Add sample volume，0.02mL V2---Total reaction volume；0.2mL=2×10-4L； W---Sample quality，g； Mr---Glucose-1-phosphate（1PG/G1P）Molecular weight；260； 500---Number of cells, in millions； D---Dilution ratio，Undiluted is 1。 /... Read More | Products contentN669983Component240 TStorageN669983AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/thaw cycle.N669983BIndex N973-N996 Primers for Illumina24×10 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit to the transposase-Products contentN669983Component240 TStorageN669983AIndex N501 Primers for Illumina240 µL-20℃. Avoid freeze/thaw cycle.N669983BIndex N973-N996 Primers for Illumina24×10 µL-20℃. Avoid freeze/thaw cycle. Products IntroductionThis kit is a companion kit to the transposase-based Rapid DNA Library Construction Kit for Illumina platform library construction. Each kit contains one N5 primer and 24 N7 primers, which can be used to prepare 24 different single-ended Index libraries. All reagents provided in the kits have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. The libraries can be used for sequencing on Illumina platforms such as HiSeq X-10/4000/2500/2000 and MiSeq. Provide your own instruments, reagents and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. DNA building kit: It is recommended to use the Century transposase method second-generation sequencing rapid DNA building kit.4. Anhydrous ethanol.5. Reaction tubes: It is recommended to use low adsorption PCR tubes with 1.5 ml centrifuge tubes;Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and library samples. Pre-experiment Preparation and Important NotesPlease centrifuge briefly before opening the cap so that the liquid collects at the bottom of the tube to avoid cross-contamination between different primers. ProcedureFor the use of the CombiVision Second Generation Sequencing Multisample Primer Kit, please follow the CombiVision Second Generation Sequencing Rapid DNA Library Kit protocol.Index N501 Primer for IlluminaIndex N901-N996 Primer for Illumina... Read More | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More |