| Description | Inquire | Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly Calcein AM /PI Double Staining Kitis utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stains viable and dead cells, respectively(Fig. 1). Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (excitationat 490 nm, emission at515 nm). Therefore, Calcein-AM only stains viable cells. On the other hand, PI, a nuclei staining dye, cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (excitation: 535 nm,emmision: 617 nm). Since both calcein and PI-DNA can be excited with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. With 545 nm excitation, only dead cells can be observed (Fig. 1). Since optimal staining conditions differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be individually determined. Please note that PI is suspected to be highly carcinogenic;careful handling is required.Required Equipment and Materials:Microscope with 490 nm excitation filter and 530 nm emission filter；CO2incubator；10 µl and 200 µl adjustable pipettes, PBSSolution A (Calcein-AM)；Solution B (PI) Storage Condition: -20oC ；Shipping Condition: blue ice.Application:Assay Procedure1)Add 2.5 µl Solution A and 12.5 µl Solution B to 5 ml PBS to prepare assay solution.*2)Wash the cell with PBS several times to remove residual esterase activity.3)Add 100uLof assay solution to200uL105~106CELLSsolution and incubate the mixture at 37oC for 15 min.4)Detect fluorescence using a fluorescence mircoscope with 490 nm excitationfor simultaneous monitoring of viable and dead cells.With 545 nm excitation, only dead cells can be observed.*The following steps may be necessary tooptimizethe suitable concentration of each reagent:1)Prepare dead cells by 10 min incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 min incubation in 70% ethanol.2)Stain dead cells with 0.1-10 µM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3)Stain dead cells with 0.1-10 µM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stainviable cells with that Calcein-AM solution to check whether the viable cell can be stained... 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 | Inquire | 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 |