| Description | Inquire | Product IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized stateProduct IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized state, it appears purple-blue and non-fluorescent, while in the reduced state, it turns into a reduction product with pink or red fluorescence, with an absorption peak of 530-560nm and an emission peak of 590nm.In the process of cell proliferation, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD in the cell increase and are in a reducing environment. The dye taken into the cell is reduced by these metabolic intermediates and cytochromes and then released outside the cell and dissolved in the culture medium, changing the culture medium from non-fluorescent indigo blue to fluorescent pink. Finally, use an ordinary spectrophotometer or fluorophotometer for detection, and the absorbance and fluorescence intensity are proportional to the number of active cells.Instructions1. Add 10µl of detection reagent to 100µl of cell suspension, and incubate in a cell incubator for 2-6 hours. The color of the medium changes from indigo blue to pink and you can proceed to the next step.2. It is recommended to use a fluorescence microplate reader for detection, the excitation light wavelength is between 530-560 nm, the emission light wavelength is 590 nm, and the relative fluorescence unit (RFU) is recorded.3. Draw a standard curve or cell growth curve: the ordinate (Y axis) is the relative fluorescence unit (RFU); the abscissa (X axis) is the cell number or time point or drug concentration.Precautions1. The appropriate density of cells can increase the detection sensitivity. For 96-well plates, we recommend seeding 100 microliters of cells per well. The cell concentration range is: 100-10,000/well for adherent cells, 2,000-50,000/well for suspension cells, and medium as a blank control. For 384-well plates, the cell concentration and seeding volume are both halved.2. The whole process should be aseptic operation, because microbial contaminants can also reduce the detection reagents and affect the experimental results.3. Pay attention to the concentration of inoculated cells and the incubation time after adding detection reagents. If the cell concentration is too high or the incubation time is too long, it will cause a secondary reduction reaction, resulting in colorlessness and disappearance of fluorescence.4. When incubating, avoid light.5. This product can use fluorescence or spectrophotometric detection, but the sensitivity of fluorescence is high, and the experimental error is small. Fluorescence detection is recommended... Read More | Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not freeze. Product Introduction：The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which canObtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and noAdditional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred toAfter coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solutionAfter washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein oneThe use of an experimental system derived from rabbits.Notes:1. Customers need to prepare their own rabbit source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), and Wash Buffer (10 x) rinse solution, please mix thoroughly.3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (rabbit) needs to be determined through preliminary experiments.6. Antibody reaction solution HRP (rabbit), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.7. The antibody dilution solution containing the first antibody can be recycled and reused once. Antibodies with low specificity and affinity are not recommended for repeated use. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.Operation steps:This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Rabbit) 100 µ Add rabbit derived primary antibody 3-10 into the centrifuge tube µ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 µ Add HRP (rabbit) antibody reaction solution into the EP tube and add 10 µ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 60 rpm for 40 minutes.6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes.7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.Example 1: Antigen 293T cell lysateA: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed Example 2 Antigen is 293T cell lysateC: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposedD: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed... Read More | Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Collection Tubes50 sets 200 setsRTS666146HCentrifuge Tubes (1.5 mL)50 EA200 EARTProductsThis kit provides a simple and rapid method for the isolation and purification of total DNA from buccal swab samples. The kit adopts a silica matrix membrane that can specifically bind DNA and a unique buffer system to adsorb DNA efficiently and specifically, and 0.5-3.5 µg of genomic DNA can be obtained from each swab, and the extracted DNA fragments are large, pure and of stable and reliable quality. It is suitable for enzyme digestion, PCR, library construction, Southern hybridization and other experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.2. If precipitation is found in Buffer GL before use, dissolve Buffer GL in a 56°C water bath.3. All centrifugation steps can be performed at room temperature.4. Sampling: Use a buccal swab to wipe the inside of the mouth 6 times, dry for 2 hours and store. To ensure that the sample is not contaminated by food or drink, do not eat or drink for 30 minutes before sampling.Procedure1. The swab of the buccal swab was cut from the rod with scissors and placed in a 2mL centrifuge tube (supplied) and 400µL Buffer GR was added.Note: For genomic DNA without RNA contamination, add 4 µL of RNase A solution at a concentration of 100 mg/ml and shake to mix.2. Add 20 µL of Proteinase K and 400 µL of Buffer GL, immediately vortex and shake for 15 seconds and mix thoroughly.Note: Mix well immediately after adding Buffer GL; do not add Proteinase K directly to Buffer GL for use.3.56°C for 10 minutes and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.4. Add 400 µL of anhydrous ethanol, vortex and shake to mix thoroughly, and centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add the solution and precipitate obtained in the previous step to the Spin Columns DS in two batches of up to 700 µL at a time into the collection tube. centrifuge the column at 12,000 rpm (∼13,400 × g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.6. Add 500 µL of Buffer GW1 to the adsorbent column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 3 minutes, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 1 minute and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.Attention:(1) If the downstream experiment is sensitive to pH or EDTA, it can be eluted with sterilized water. The pH value of the eluent has a great influence on the elution efficiency. If the eluent is made of water, the pH value should be 7.0-8.5 (the pH value of water can be adjusted to this range by using NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2) For long-term storage, it is recommended to elute with Buffer GE and store at -20°C... Read More | Component Description T665563Component50 TStorageApplicationT665563AVNTR3820 1 mL-20℃. Avoid freeze/thaw cycle.High resolution 3-lite VNTR detectionT665563BVNTR41201 mL-20℃. Avoid freeze/thaw cycle.High resolution 3-lite VNTR detectionT665563CVNTR32321 mL-20℃. Avoid freeze/thaw Component Description T665563Component50 TStorageApplicationT665563AVNTR3820 1 mL-20℃. Avoid freeze/thaw cycle.High resolution 3-lite VNTR detectionT665563BVNTR41201 mL-20℃. Avoid freeze/thaw cycle.High resolution 3-lite VNTR detectionT665563CVNTR32321 mL-20℃. Avoid freeze/thaw cycle.High resolution 3-lite VNTR detectionT665563DMarkerⅠ300 µL-20℃. Avoid freeze/thaw cycle.DNA Molecular Weight Standard IT665563EMarkerⅡ250 µL-20℃. Avoid freeze/thaw cycle.DNA Molecular Weight Standard IIProduct IntroductionThis kit is a genotyping product for human Mycobacterium tuberculosis based on the latest research progress in molecular epidemiology1) and optimized by process. It utilizes variable-number tandem repeats (VNTR) polymorphisms in the Mycobacterium tuberculosis genome for genotyping to differentiate clinical strains, and is a powerful tool for studying the molecular epidemiology of Mycobacterium tuberculosis and monitoring the status of tuberculosis transmission. Compared with other existing Mycobacterium tuberculosis VNTR typing systems based on the VNTR principle, this typing system has a stronger ability to discriminate strains prevalent in China1,2,3), and is therefore particularly suitable for the needs of Chinese users.By carefully optimizing the primer sequences of each PCR reaction and the composition of the premixed reaction solution, this product has a strong anti-interference power. Compared with the user's own reagents, this product significantly improves the signal intensity of specific bands and reduces the appearance of non-specific bands when using crude templates (boiling bacterial solution), which makes the experimental operation easier and quicker, and at the same time, improves the success rate of the test. The premixed reaction solution is chemically stable and can effectively withstand repeated freezing and thawing (10 times) and a longer period of time (one week) at room temperature, which is better adapted to the user's need for flexibility in the detection work.This kit is a companion product to the TB Genotyping Kit VNTR-9. For samples identified as clustered or identical strains by the VNTR-9 kit, this product can be used for finer further typing identification if necessary. The three high-resolution detection sites VNTR3820, VNTR4120 and VNTR3232 in this product can be used in combination with the nine detection sites in the VNTR-9 to increase the resolution index (Hunter-Gaston index (HGI) to 0.9931).References1) Luo T et al. Development of a hierarchical variable-number tandem repeat typing scheme for Mycobacterium tuberculosis in China. PLoS One. 2014 Feb 25. 9(2)2)Sun G et al. Discriminatory potential of a novel set of Variable Number of Tandem Repeats for genotyping Mycobacterium marinum. Vet Microbiol. 2011 Aug Vet Microbiol. 2011 Aug 26;152(1-2)3) Zhang L et al. Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains of Mycobacterium tuberculosis in Shanghai, China. FEMS Microbiol Lett. 2008 May;282(1):22-31.matters needing attention1.This product is a companion to the TB genotyping kit VNTR-9. The strains to be tested should be tested by VNTR-9 typing test first, and then use this product for testing. And the results of this product should be integrated and analyzed with the results of VNTR-9.2.To avoid contamination, it is recommended that the preparation of the organisms be done within a different location than the preparation of the PCR Mix and that different pipettes be used.3.Care should be taken at all stages of sample DNA collection, extraction and amplification to ensure proper labeling and to prevent cross-contamination between different samples.4.Commonly used reagents and consumables need to be autoclaved before experimentation.5.Each tube of PCR Mix contains different primers and cannot be mixed. It can be dispensed into different amounts at once according to the experimental needs to avoid repeated freezing and thawing.6.To avoid splashing the reaction solution when opening the reaction tube, centrifuge briefly before opening the cap and collect the liquid at the bottom of the tube. In case of accidental splashing on gloves or table, change gloves immediately and wipe the table with 75% alcohol or dilute acid.7.Be careful not to cross-contaminate the PCR Mix when aspirating, and it is recommended that the pipette tip be wiped with 75% alcohol 2 times before taking Mix each time.8.Pre-experiment preparation: 1×TE buffer (PH=8.0), 0.5×TBE buffer, agarose, ethidium bromide (EB), normal PCR instrument, DNA electrophoresis equipment and gel imager, 0.2 ml PCR reaction tubes, octuplex or 96-well PCR tubes, pipettes of different sizes: 0.5-10 µl and 20-200 µl.Operation steps1. DNA template preparation:1.1. scrape a small amount (1-2 inoculation loops) of sample from solid medium, resuspend in 100ul TE and inactivate at 80°C for 30 minutes.1.2. The inactivated strain was taken out of the P3 laboratory as follows:Boil at 100°C for 10 minutes (be careful to avoid bursting the cap of the EP tube during boiling to avoid letting water into the tube), place immediately on ice for 2 minutes, centrifuge at 12,000 rpm (~13,400 × g) for 10 minutes, take the supernatant and place in another sterile EP tube, label it, and store at -20°C.2. Testing procedures:2.1. Remove the TB Genotyping Kit HV-3, allow the liquid to equilibrate to room temperature, mix by shaking slightly 3-4 times, and then centrifuge at 12,000 rpm (~13,400 x g) for 5 seconds to allow the capped liquid to fall back into the tube.2.2.Three-locus VNTR typing: strains with identical results at 12 loci need to be further VNTR typed, i.e., the following four loci are added for comparison.1）PCR amplification: the reaction system was 20 µl. 19 µl of PCR Mix of VNTR3820, VNTR4120, and VNTR3232 were added to each PCR tube, 1 µl of DNA template was added, and mixed well.2）Amplification conditions:3) Gel preparation and electrophoresis:a: Notes:Important! Positive (H37Rv strain DNA) and negative controls (deionized water) need to be set up for each experiment.Key! This experiment is based on agarose gel electrophoresis to interpret the genotype of VNTR locus, therefore, in order to make the results accurate, it is necessary to follow the unified standard operation in this step of electrophoresis, and the following points should be noted:a-1: The comb used for glue making is 18 holes.a-2: The two wells on the left and right sides of the gel were discarded due to the tendency to distort the bands during electrophoresis, affecting the interpretation of the results, or a negative control was spotted in one of the wells. The remaining 16 wells were divided into 12 samples, 3 DNA Markers and 1 positive control. The order of spotting was "1, 2, M, 3, 4, 5, 6, M, 7, 8, 9, 10, M, 11, 12, H37Rv", the numbers represent samples, and M represents DNA Marker.a-3: When PCR amplification products are subjected to the first electrophoresis and Marker I is used, the gel concentration is 1%, the voltage is 150 V, and the time is 100-120 min.a-4: If the amplification product fragment is too large (>1000bp) and needs to be electrophoresed again and Marker II is used, the gel concentration is 0.8%, the voltage is 150V and the time is 150 minutes.b: Gluing as well as the electrophoresis process:PCR amplification products were electrophoresed using a 1% agarose gel.To prepare 1% agarose gel, 12×12 cm gel tray was used to make the gel, each gel was 80 ml.b-1: Weigh 0.8g of agarose, add 80ml of 0.5×TBE, weigh it on the balance and put it into the microwave oven, heat it on high for 2-3 minutes to make the agarose dissolve completely, shake it well, and observe it as a homogeneous and transparent solution without particles, then weigh it again on the balance and make up the appropriate amount of double-distilled water to keep the concentration of the gum unaffected.b-2: When the melted gel was cooled to about 55°C add 4 µl of ethidium bromide (10ug/ml) and gently swirl to mix well. The gel was made with an 18-tooth comb and the warm gel was poured into a 12 × 12 cm gel tray.b-3: Allow the gel to completely set (40 minutes at room temperature), carefully pull out the comb, remove the tray, and place it in the electrophoresis tank. Add 0.5× TBE buffer to the electrophoresis tank, not exceeding the gel surface by 1-2mm.b-4: Sample electrophoresis: add 12 samples to each gel (the topmost wells are not sampled), add 3-5µl PCR products to each well, and at the same time add three 5µl DNA MarkerⅠ to each gel. The voltage is 150V and the electrophoresis time is 100-120 minutes. This step is the key to the accuracy of the final readings of each point, and needs to be operated uniformly according to this standard.b-5: Some loci have amplification products greater than 1000bp in clinical strains, and these amplification products were then electrophoresed using 0.8% agarose gel, with DNA Marker II added as a control for the band size, voltage 150V, electrophoresis time 150 minutes.4) Results display:5) Analysis of results:a. If the genotypes of the three highly variable loci are also the same in different strains, they can be identified as clustered strains;b. If the high variant readings are highly similar, i.e., only 1-2 high variant sites are different, they need to be combined with epidemiologic data to identify if they are clustered strains;c. If all 3 high variant loci are genotypically discordant, identify as a single strain.Appendix 1: Rules for reading VNTR lociAppendix 2: VNTR locus repeat unit readout table... Read More |