| Description | Inquire | Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-ethynyl-2 '- deoxyuridine) is a pyrimidine analog that integrates into the DNA duplex during DNA synthesis. Edu detection is based on the "click" reaction. A copper catalyzed azide reacts covalently with alkynes to form covalent bonds. In this kit, edu contains alkynes, Aladdin ® 488 / 555/594/647a azide dyes contain azide compounds. The edu labeling proliferation of click method is rapid and effective, and easy to use. BrdU method requires DNA denaturation (such as acid denaturation, thermal denaturation or digestion with DNase) to expose BrdU, so as to facilitate BrdU antibody binding; The edu method only needs paraformaldehyde fixation and Triton X-100 penetration to make the detection reagent enter the cells, and only a small amount of azide dye is needed to label the integrated edu very effectively. This kit contains all components required for edu method detection, and can be used for proliferation detection of cultured cells in vitro.Component:Product parameters：555/565 nm；Instruction： Experimental materials (self provided). 10 mM PBS, pH 7.2-7.6. 4% paraformaldehyde fixing solution (in PBS)Propensive reagent (0.5% Triton X -100 in PBS). 2 mg/mL glycine solution (in ddH2O). 3% BSA in PBS, pH 7.2-7.6. 1% BSA in PBS, pH 7.2-7.6. ddH2O. 96/24/12/6 well culture plate or dishFluorescence microscopy detection method1. Cell cultureTake logarithmic growth stage cells and inoculate them into a 96 well plate with 4 × 103-1 × 105 cells per well (the number and density of cells can be adjusted according to cell size, growth rate, and specific requirements of experimental treatment), and culture until normal growth stage.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU marking(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations, such as 10-50, should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash the cells twice with a 3% BSA washing solution after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, remove the culture medium. Wash cells twice with 1X PBS for 5 minutes each time to remove EdU residues that have not been incorporated into DNA. Cells with weak adhesion can reduce cleaning intensity. Join 50 µ After incubating at room temperature for 20 minutes with 4% paraformaldehyde fixative, remove the fixative.(2) Add 50 to each hole µ L 2 mg/mL glycine solution, incubate at room temperature for 5 minutes, and neutralize the remaining fixed solution.(3) At a rate of 100 per hole µ Wash cells twice with 3% BSA.(4) Remove the washing solution and add 100 µ L 0.5% Triton X -100, incubate at room temperature for 10 minutes.5. EdU detectionNote: Each sample in this reference step uses 100 µ The working fluid of L can be adjusted by users according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer (component C): Dilute component C 10 times with ddH2O.(2) Configure 5 x Click iT EdU buffer additives (component E): add 300 µ Mix L of ddH2O into a 30 mg E component tube (final concentration of 100 mg/mL) until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio, and prepared into a 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 1.Table 1 Click it working fluidReaction componentsTaking the sample size of 10 holes as an example1×Click-iT EdU Reaction buffer855 µLCuSO4 (Component D)40 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Remove penetration enhancer, 100 per well µ Wash twice with 3% BSA washing solution of L.(6) Add 100 to each hole µ L Click iT working solution, evenly covering cells.(7) Incubate at room temperature in dark for 30 minutes.(8) Remove Click-iT working fluid and add 100 µ After washing cells twice with 3% BSA, remove the washing solution and add 100 µ L PBS keeps cells moist. If there are no other special requirements, photography analysis can be carried out.6. DNA re staining (optional)(1) Using 100 µ Wash the cells once with PBS and remove the washing solution.(2) Dilute Hoechst 33342 (component F) 2000 times with PBS.(3) Add 100 to each hole µ Incubate L 1 x Hoechst 33342 solution at room temperature in dark for 15-30 minutes.(4) Remove Hoechst 33342 solution and use 100 µ Wash cells twice with PBS.7. Imaging and analysisIt is recommended to take fluorescence microscopy photos immediately after staining is completed for observation; If conditions permit, please store in a dark and moist environment at 4 ° C for 3 days before taking photos. Flow cytometry detection method1. Cell cultureInoculate 1 × 105~3 × 106 cells per well into a 6-well plate.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU labeled cells(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours. The time of EdU incubation of cells can be directly used as an indicator for measuring cell DNA synthesis, and the choice of time point and incubation time depend on the cell growth rate. Pulse labeled cells incubated with brief EdU can be used to study cell cycle dynamics.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations such as 10-50 should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash cells twice with 1% BSA after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, collect cells, add 1 mL of PBS to each tube to clean the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove EdU residue that has not been added to DNA.(2) Add 1 mL of 4% paraformaldehyde fixative to each tube to resuspend cells.(3) Incubate at room temperature for 20 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(4) Add 1 mL of 2 mg/mL glycine to each tube and incubate for 5 minutes. Neutralize the remaining fixed solution, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of PBS to each tube for cleaning once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(5) Add 1mL of 0.5% Triton X-100 osmotic enhancer to each tube and resuspend cells. Incubate at room temperature for 10 minutes.5. EdU detectionNote: For 6-well plate samples, reference can be made to 1 mL of working solution per well. Users can adjust the dosage according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer: Dilute component C 10 times with ddH2O.(2) Prepare 5 x Click iT EdU buffer additives (component E): Add 300 µ L ddH2O to 30 mg of component E in a test tube (final concentration 100 mg/mL), mix well until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio to form 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive storage solution with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 2.Table 2 Click it working fluidReaction componentsVolume of liquid required for a single reaction1×Click-iT EdU Reaction buffer875 µLCuSO4 (Component D)20 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Soak at 1000 rpm for 5 minutes, discard the supernatant, remove the enhancer, add 1mL of 1% BSA washing solution to each tube and wash twice. Soak at 1000 rpm for 5 minutes, discard the supernatant.(6) Add 1 mL of Click iT working solution to each tube and mix well.(7) Incubate at room temperature in dark for 30 minutes.(8) Soak at 1000 rpm for 5 minutes, discard the staining reaction solution, add 1% BSA to each tube to wash the cells twice, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells again with 1 mL of 1% BSA (the volume of resuspend cells can be adjusted according to the number of cells), and detect with a flow cytometer.Note: If other biomarker tests are required, please refer to step 4.6. Intracellular antigen labeling (optional steps)(1) Add antibody working solution and mix well.(2) Under dark conditions, incubate antibodies at appropriate temperature and time.7. Flow detection and analysis:(1) It is recommended to conduct flow cytometry testing immediately after dyeing is completed; If conditions are limited, please store in a dark place at 4 ℃ for testing, but it should not exceed 3 days.(2) It is recommended to test the number of cells up to one million levels as much as possible. If the number of cells is small, the number of cells tested can be adjusted to 100000 levels starting from the experiment. For cases where the cell yield is too low (just to the level of ten thousand), it may not be conducive to making a flow chart. Therefore, the cleaning frequency in step 5 (8) can be appropriately reduced.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 3. click it edu buffer additive solution should be prepared and used immediately to ensure the best results. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell proliferation detection (cell imaging flow universal)... Read More | B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets B669951 Component 50T Storage B669951A Buffer ATL 15 mL RT B669951B Buffer AL 15 mL RT B669951C Buffer AW1 (concentrate) 13 mL RT B669951D Buffer AW2 (concentrate) 15 mL RT B669951E Buffer EB 15 mL RT B669951F Proteinase K 1.25 mL RT B669951G Spin Columns DM with Collection Tubes 50 sets RTProductsThis kit is suitable for extracting high purity total DNA from Gram-negative and Gram-positive bacteria. 106-108 cells can be processed at a time, and up to 20 µg of total DNA can be obtained within one hour without the need for toxic solvents such as phenol or chloroform, and without the need for ethanol precipitation. The optimized buffer system enables the DNA in the lysate to be efficiently and specifically bound to the silica matrix centrifugal adsorption column, while other contaminants can flow through the membrane, and the inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing step, and finally washed off with low-salt buffer or water, so that high-purity DNA can be obtained.The purified DNA can be used for downstream experiments such as digestion, PCR, Real-Time PCR, library construction, Southern Blot and molecular labeling, molecular labeling and other downstream experiments. Self-contained reagents: anhydrous ethanol; Enzymatic Lysis Buffer is required for extraction of Gram-positive bacteria.Enzymatic Lysis Buffer was prepared by 20 mM Tris, pH 8.0; 2 mM Na2-EDTA, pH 8.0; and 1.2% Triton X-100. 121°C sterilization for 20 minutes, and the appropriate amount of Lysozyme was added at a final concentration of 20 mg/ml. Pre-experiment Preparation and Important Notes1. Add 1.25ml Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. If extracting genomes from bacterial cultures with high accumulation of secondary metabolites or thick cell walls, it is recommended that samples be collected early in the logarithmic phase.4. 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.5. Before use, please check Buffer GTL and Buffer GL for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer GL and Buffer GTL in a 56℃ water bath.6. If the downstream experiments are sensitive to RNA contamination, 4µl of DNase-Free RNase A (100mg/ml) can be added before adding Buffer GL. RNase A is not provided in this kit.If the extracted samples are Gram-positive bacteria, customers need to prepare their own Enzymatic Lysis Buffer to treat the bacteria, which requires the use of Lysozyme (lysozyme) at a concentration of 20 mg/ml, which is not provided in this kit.Procedurei Extraction of genomic DNA from Gram-negative bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180 µl Buffer GTL to the precipitate and shake to resuspend the bacteria.3. Add 20 µl of Proteinase K, vortex and mix well, incubate at 56°C until the solution becomes clear, and invert or shake the centrifuge tube at intervals during the incubation to disperse the sample.Note: If RNA removal is required, add 4 µl of RNase A solution at a concentration of 100 mg/ml after the above steps are completed, shake to mix, and leave for 5-10 minutes at room temperature.4. Add 200µl Buffer GL and mix well with vortexing and shaking. Add 200µl of anhydrous ethanol and mix well with vortexing and shaking.Centrifuge briefly so that the solution on the walls of the tube collects at the bottom.Note: 1) If multiple samples are manipulated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and then added together, shaking to mix.2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in step 4 (including the precipitate formed) to the Spin Columns DM in the collection tube, or if the solution cannot be added all at once, transfer it several times. centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption 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 return the adsorption column to the collection tube.7. Add 500 µl of Buffer GW2 to the adsorption 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 adsorption 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 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorbent column at room temperature for several minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube, add 50-200 µl Buffer GE to the middle part of the adsorption column overhanging the center of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃. note: 1) If the downstream experiments are sensitive to the pH or EDTA, the elution can be done with sterilized water. The pH of the elution solution has a great influence on the elution efficiency. If water is used as the elution solution it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 9 can be re-spiked onto the adsorbent membrane and step 9 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃.i. Extraction of genomic DNA from Gram-positive bacteria1. Take 1-5 ml of bacterial culture (106-108 cells, maximum 2×109 cells) and put it into a centrifuge tube (provided), centrifuge it at 12,000 rpm (~13,400×g) for 1 minute, and aspirate the supernatant as much as possible.2. Add 180µl Enzymatic Lysis Buffer (self-provided) to resuspend the bacteria.Enzymatic Lysis Buffer is prepared as described in the Self-Prepared Reagents section in the front of the manual.3. Incubate at 37°C for 30 minutes.4. Add 20µl Proteinase K and mix well. Add 200µl of Buffer GL and mix well with vortexing and shaking.Note: Do not add Proteinase K directly to Buffer GL.Incubate at 5.56°C for 30 minutes.Note: 1) If desired, incubation at 95°C for 15 minutes will inactivate the pathogen, but 95°C incubation will cause some DNA degradation.(2) If RNA removal is required, add 4µl of RNase A solution at a concentration of 100mg/ml after the above steps are completed, shake and mix well, and leave for 5-10 minutes at room temperature.6. Add 200µl of anhydrous ethanol and mix well with vortex shaking.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.7. Add all of the solution obtained in step 6 (including the precipitate formed) to the Spin Columns DM that have been loaded into the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and put the column back into the collection tube.8. Add 500 µl of Buffer GW1 to the adsorption 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 adsorption column back into the collection tube.9. Add 500 µl Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge the column at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 9 can be repeated if further DNA purity is required.10. Centrifuge at 12,000 rpm for 2 minutes 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; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).11. Place the adsorption column in a new centrifuge tube (self-provided), add 50-200 µl of Buffer GE to the center of the adsorption column overhanging the center of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20℃.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Incubation at room temperature for 5 minutes prior to centrifugation increases yield.3) Re-elution with an additional 50-200 µl Buffer GE or sterilized water can increase the yield.4) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 11 can be re-spiked onto the adsorbent membrane and step 11 repeated; if the elution volume is less than 200 µl, the final concentration of DNA can be increased, but the total yield may be reduced. If the amount of DNA is less than 1 µg, elution with 50 µl Buffer GE or sterilized water is recommended.(5) DNA stored in water will be affected by acidic hydrolysis. For long-term storage, it is recommended to elute with Buffer GE and store at -20℃... Read More | N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917 Component 1 mL 5 mL Storage N665917A 2×SYBR qPCR MasterMix 1 mL 5×1 mL -20℃. Avoid freeze/ Thaw cycle. N665917B qPCR Primer Mix 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917C DNA Standard A 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917D DNA Standard B 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917E DNA Standard C 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917F DNA Standard D 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917G DNA Standard E 100 µL 500 µL -20℃. Avoid freeze/ Thaw cycle. N665917H 50×High ROX 40 µL 200 µL -20℃. Avoid freeze/ Thaw cycle.Product IntroductionThis is a dye-based (SYBR Green I) qPCR NGS library quantification kit for cfDNA, which provides the reaction mixture, DNA primer mixture, standards, and sample dilutions required for the qPCR process, making it a complete reagent system that is easy and convenient to use. The fluorescent dye SYBR Green I contained in the reaction mixture binds to all double-stranded DNA. The kit uses a new chemically modified high-efficiency hot-start polymerase, the activation of the enzyme needs to be incubated at 95 ℃ for 10 min. the product is highly specific, high amplification efficiency, the length of the standard in the kit (about 270bp) is comparable to the average length of the cfDNA NGS libraries (250-300bp), which is able to quickly and accurately quantitate the concentration of the constructed cfDNA libraries. quantification.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.NOTE: High Rox and Low Rox are formulated as described in Method of Use 2.Applicable scopeThis product is designed for the absolute quantification of the concentration of Illumina platform second generation sequencing libraries. The end of the library contains Illumina P5 and P7 microarray binding sequences, the length of which does not exceed 1kb, and the concentration is not less than 0.02pM can be used for quantitative experiments. The qPCR Primer Mix provided in the kit contains the following two primer sequences:Primer 1:5'-AAT GAT ACG GCG ACC ACC GA-3' Primer 2: 5'-CAA GCA GAA GAC GGC ATA CGA-3'The primer sequence can be used in advance to confirm whether the library can be amplified by that primer pair.UsageAmplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.01-60 pM. 4°C on ice was set aside.qPCR reaction system preparationThe desired cryopreservation reagent is pre-melted completely and mixed by inverting several times before preparation, then centrifuged briefly and set aside.The base reaction system for 20 µl was as follows:Reagent20 µl Reaction system2×SYBR qPCR MasterMix10 µlqPCR Primer Mix0.8 µlTemplate4 µlddH₂O5.2 µlDescription: High Rox model: 1 µl High Rox per 50 µl of reaction system; Low Rox model: 1 µl High Rox per 500 µl of reaction system.Prepare a sufficient amount of reaction system mixture according to the need, mix well and add to the reaction wells in a volume of 16 µl per well, add the same volume of TE to the blank control, and then add the prepared standards and diluted samples to the corresponding reaction wells in a volume of 4 µl/well. It is recommended to use 20 µl reaction system, if you need to carry out a smaller system reaction, the system components can be reduced in equal proportion.3.qPCR reaction programIf the average length of the library is greater than 700bp, the annealing/extension time should be increased appropriately.Refer to the specific instrument setup program for dissolution curves.data analysisStandard curve productionThe standard curve was plotted according to the data processing Excel sheet. The correlation coefficient R2 of the standard curve should be not less than 0.99, and the slope should be located between -3.1 and -3.6 when the Ct value is the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard ConcentrationDNA Standard A60 pMDNA Standard B6 pMDNA Standard C0.6 pMDNA Standard D0.06 pMDNA Standard E0.006 pMLibrary Concentration CalculationsThe difference in Ct between the three replicate wells of the experiment should be no more than 0.2, otherwise the invalid data should be deleted or the experiment should be repeated. Do not use the Ct outside the valid Ct range of the standard curve to calculate the concentration of the diluted libraries. Please refer to the data processing Excel of this product for the specific library concentration calculation method.matters needing attentionThese instructions should be read in detail before testing. It should be carried out by personnel with specialized experience or qualified by training.Mix gently by turning up and down, avoid foaming as much as possible, and centrifuge for a short time before use.Avoid repeated freezing and thawing of this product; repeated freezing and thawing may degrade product performance.When preparing reaction solutions, use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More | Product content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligationProduct content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligation of DNA sticky or flush ends in 5 minutes at room temperature (25°C). The kit contains Quick T4 DNA Ligase and 2×Quick Ligation Reaction Buffer optimized for fast and efficient DNA ligation.The ligation efficiency of Quick Ligation is equivalent to 1 hour of conventional ligation with T4 DNA Ligase. The Quick Ligation products can be used directly in routine bacterial transformation experiments.matters needing attention1. This kit enables most of the linkage reactions to reach the reaction endpoint within 5 minutes or less at 25°C. Increasing the reaction time will not enhance the reaction efficiency. If you use the rapid connection reaction after 1 hour, the conversion efficiency will be significantly reduced; if the rapid connection reaction at 25 ℃ overnight, the conversion efficiency will drop to 75%.2. 2×Quick Ligation Reaction Buffer contains ATP, which should be thawed on ice and mixed thoroughly before use. It is recommended to freeze the buffer in small tubes for the first time, so as to avoid repeated freezing and thawing, which will affect the efficiency of DNA ligation.3. Since T4 DNA Ligase contains glycerol, which is sticky and easy to hang on the wall, it is recommended to collect the liquid to the bottom of the tube by centrifugation for a short period of time before use, and the tip of the lance should not go too deep into the liquid surface when taking samples to avoid sticking to the tip of the lance and causing losses.4. If the quick ligation product is used for electrotransformation, the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation, and it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation.Usage1. The reaction solution was prepared according to the following system:*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.5. Heat shock the ligation product to transform 50 µl of receptor cells or take 1-2 µl of ligation product to electroshock transform 50 µl of receptor cells.Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation... Read More |