| Description | 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 | Inquire | FFPE DNA/RNA KitFixed Tissue DNA/RNA Extraction Kit Catalog number: F666120 (50 preps)Storage conditions: DNase I and 10×Reaction Buffer -20℃, Spin Columns DF and Spin Columns RS can be stored at room temperature for 2 months, 2-8℃ for 1 year, the rest of the components are stored FFPE DNA/RNA KitFixed Tissue DNA/RNA Extraction Kit Catalog number: F666120 (50 preps)Storage conditions: DNase I and 10×Reaction Buffer -20℃, Spin Columns DF and Spin Columns RS can be stored at room temperature for 2 months, 2-8℃ for 1 year, the rest of the components are stored at room temperature (15-30℃).Products Content:Products IntroductionThis kit is suitable for the effective purification of genomic DNA and total RNA from paraffin-embedded tissues, using specially optimized deparaffinizing agents and lysates to release DNA and RNA from tissue section samples, without the use of the organic reagent xylene, and without the need for overnight operation; the digested samples are incubated at higher temperatures to remove inhibitors caused by cross-linking, which can effectively improve nucleic acid yields and purity; and an optimized buffer system allows nucleic acids in the lysate to bind specifically to the adsorbent membrane, and inhibitors are effectively removed by a two-step rinsing procedure. The optimized buffer system enables the nucleic acids in the lysate to bind specifically to the adsorbent membrane, and the inhibitors are effectively removed by a two-step rinsing step, and finally eluted with low-salt buffer or water to obtain high purity DNA and RNA, and at the same time, equipped with a high-efficiency microsorbent column, the volume of the elution can be as low as 20 µl. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, and so on. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, second-generation sequencing, pharmacogenomics research and blot analysis.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes 1. After obtaining the sample, fix the sample as soon as possible, the fixation time of 14-24 hours is appropriate, too long a period of time will easily lead toDNA and RNA breaks, affecting downstream experiments. If the formaldehyde fixation time is too long or the sample is stored for too long(>1 year) is prone to compromise DNA integrity and failure to amplify long fragments.2. Ensure that samples are thoroughly dehydrated prior to embedding; residual formalin will inhibit Proteinase K action.3. Add 1.25 ml of Proteinase K Storage Buffer to Proteinase K to dissolve it, and store at -20℃. Do not leave the prepared Proteinase K at room temperature for a long period of time to avoid affecting its activity.4. Anhydrous ethanol should be added to Buffer RW2, Buffer GW1 and Buffer GW2 according to the label instructions on the vials before first use.5. Check Buffer GTL, Buffer GL and Buffer DS for crystallization or precipitation prior to use; if crystallization or precipitation occurs, redissolve Buffer GTL, Buffer GL and Buffer DS in a 37°C water bath.6. Preheat the water bath or thermostatic mixer to 56°C before starting the experiment.7. Use an ambient temperature centrifuge or set the centrifuge temperature to 25°C. Temperatures below 15°C may result in clogging of the adsorption column.8. To prevent RNase contamination, the following should be observed:1) Use RNase-free plastics and tips to avoid cross-contamination.(2) Glassware should be dry baked at 180°C for 4 hours before use, plasticware can be soaked in 0.5 M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.3) RNase-free water should be used to prepare the solution.(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.procedureParaffin-embedded samples1. Trim off excess paraffin from the tissue block to expose the tissue and cut into 5-10 µm slices.2. Place approximately 1 x 1 cm2 slices (1-5 slices in total) in a centrifuge tube (supplied), add 500 µl of Buffer DS and vortex for 10 s. Briefly centrifuge the sample to the bottom of the tube. Centrifuge briefly to collect the sample at the bottom of the tube, incubate at 56°C for 3 minutes, remove from the water bath and allow to cool to room temperature before proceeding.Note: If the surface of the sample is exposed to air, discard the initial 2-3 slices without using them.3. Centrifuge at 12,000 rpm for 2 minutes and carefully discard the supernatant thoroughly without aspirating the precipitate. The residual dewaxing solution can be carefully removed with a small tip (10 µl).4. Add 180 µl of Buffer GTL and 20 µl of Proteinase K to the above tube and mix well with vortexing.5. Incubate at 56°C for 15 minutes, then place on ice for 3 minutes. Centrifuge at 12,000 rpm for 15 minutes at room temperature.6. Transfer the supernatant to a new 1.5 ml centrifuge tube for RNA extraction, taking care not to aspirate undigested tissue. Use the precipitate for DNA extraction. RNA extraction7. Take the supernatant obtained in step 6 and incubate at 80°C for 15 minutes.8. Add 320 µl of Buffer GL, mix by vortexing and shaking, then add 720 µl of anhydrous ethanol and mix immediately by vortexing and shaking.9. Add all of the resulting solution to the Spin Columns RS in the collection tube; if the solution cannot be added all at once, it may be transferred in several passes. centrifuge the column at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and place the column back into the collection tube. Note: If the columns are clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.Optional step: If genomic DNA is to be removed, the following steps can be followeda. Add 350 µl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and place the column back into the collection tube.b. Preparation of DNase I mixture: Take 52 µl of RNase-Free Water and add 8 µl of 10×Reaction to it.Buffer and 20 µl DNase I (1 U/µl), mix well, and prepare a final volume of 80 µl of reaction solution.c. Add 80 µl of DNase I Mix directly to the adsorption column and incubate at 20-30°C for 15 minutes.d. Add 350 µl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.Add 500 µl of Buffer RW2 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.11. Repeat step 10. centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the column at room temperature for 5 minutes.minutes to dry thoroughly.12. Place the column in a new RNase-free centrifuge tube and add 20-50 µl to the center of the column.RNase-Free Water, left at room temperature for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and collected RNA solution, the-80°C for storage.DNA extraction7. Take the precipitate obtained in step 6 and add 180 µl Buffer GTL and 20 µl Proteinase K to the precipitate. VortexResuspend the precipitate for 15 seconds.8. Incubate at 56°C for 1 hour until the sample is completely dissolved. 90°C for 1 hour.Add 200 µl Buffer GL, vortex and shake to mix and then add 200 µl anhydrous ethanol, vortex and shake to mix thoroughly. Centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube. Add all of the solution from step 9 to the Spin Columns DF in the collection tube, or transfer the solution in several passes. centrifuge at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and return the column to the 10. collection tube.Note: If the adsorption column is clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.11. Add 500 µl of Buffer GW1 to the adsorbent column and 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.12. Add 500 µl of Buffer GW2 to the adsorbent column and centrifuge at 12,000 rpm for 1 minute. Pour off the waste liquid from the collection tube and place the column back into the collection tube.Note: Step 12 may be repeated if further purity is required.13. 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 5 minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue will affect the subsequent enzymatic reaction. 14. Place the column in a new 1.5 ml centrifuge tube, add 20-50 µl Buffer EB to the center of the column, leave at room temperature for 5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃... Read More | Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of plasma membrane to reflect cell viability. The kit can be used for fluorescence microscopy, flow cytometry, microplate reader and other fluorescence detection systems. This kit can be applied to most eukarYOtic mammalian cells, including some tissues with adherent nuclei, but it is not applicable to fungi and yeast. Compared with trypan blue, the kit is faster, safer and more sensitive.Component： Product parameters：Calcein am: ex/em = 494 / 517 nm; Ethd-i: ex/em = 528 / 617 nm (bound DNA)Usage:Fluorescence microscopy detection1. Prepare working fluidPreparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Remove the original solution of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium, vortex well. The above working solution can be directly used for cell staining.Note: The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. The concentration selection of Calcein AM and EthD-I varies depending on the type of cell used, with a recommended concentration range of 0.1-10 µ M.2. Prepare cells and conduct experiments(1) For adherent cells, they can be washed 2-3 times with 1 × PBS before staining. For suspended cells, centrifuge at room temperature of 250-1000 × g for 5 minutes and collect cells for staining.(2) Wash the cells thoroughly 2-3 times with 1 × PBS to remove residual esterase activity.(3) For adherent cells, add sufficient amount of Calcein AM/EthD-I staining solution. For suspended cells, add an appropriate amount of staining solution to control the cell density between 1-5 × 105/mL.(4) Incubate at room temperature in dark for 15-20 minutes (if the working solution concentration is high or the incubation temperature is high, the incubation time should be appropriately reduced).(5) Observe the labeled cells under a fluorescence microscope.Flow cytometry detection1. Remove the reagent and restore it to room temperature.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Take out the original solution of Calcein AM and EthD-I, and restore to room temperature. Add 20 µ L 2 mMEthD-I and 5 µ Vortex mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium. The working fluid can directly stain cells.3. Wash cells thoroughly 2-3 times with 1 × PBS.4. Suspend cells with 0.5 mL of staining solution and control the cell density to 1-5 × 105/mL.Note: It is recommended to prepare two additional cell samples, each containing only one dye (Calcein AM and EthD-I), for compensatory regulation of flow cytometry single staining; Prepare another cell sample containing only buffer solution (which should be consistent with the buffer used to prepare Calcein AM and EthD-I detection working solutions) as a negative control for flow cytometry analysis.5. Incubate at room temperature in dark for 15-20 minutes.6. Within 1-2 hours, cell activity was detected by flow cytometry. Calcein AM can be excited by a 488 nm laser, with fluorescence emission spectra detected at around 530 nm and EthD-I emission spectra at around 610 nm.Note: When using the cell circle gate, attention should be paid to excluding cell debris and using a single staining tube to regulate compensation. Double staining tube flow cytometry should obtain two relatively independent cell populations: a live cell population displaying green fluorescence and a dead cell population displaying red fluorescence.ELISA reader detection1. Cultivate an appropriate amount of adherent or suspended cells in a 96 well black ELISA plate.Note: Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution:Remove the original solutions of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM 10 mL PBS or other serum-free buffer or culture medium, vortex well.Note: (1) 10 mL of staining solution is sufficient to stain a 96 well plate, and the volume of the staining solution can be adjusted according to experimental needs. The concentrations of Calcein AM and EthD-I can range from 0.1 to 10 µ Explore between M.(2) The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. EthD-I working solution can be stored at -20 ℃ for at least one year.3. Wash the cells thoroughly with 1 × PBS to remove residual esterase activity. For adherent cells, add 100 to each well µ Wash cells with PBS. For suspended cells, add 100 µ Resuspend cells with L PBS and centrifuge to remove the supernatant. Repeat the above operation.4. Add 100 to each hole µ L PBS.5. Add 100 to each hole µ L staining solution, making the total volume of each well 200 µ L. The final concentration of Calcein AM is 1 µ M. The final concentration of EthD-I is 2 µ M. Gently shake the culture plate to evenly cover the cells with the liquid.Incubate at room temperature in dark for 30-45 minutes.Note: The optimal incubation time varies for different cells, with 30 minutes as the initial incubation time. Subsequently, the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect.7. Enzyme reader detection. When the ELISA reader is set to fluorescein, it can detect Calcein AM; When the ELISA reader is set to rhodamine or Texas Red, EthD-I can be detected. Select the optimal emission and excitation wavelengths based on spectral characteristics.Note: By comparing the relative fluorescence values (RFU) measured between the sample group and the control group, the changes in the number of dead and live cells can be obtained. Another method of data analysis is also provided below.The following method can calculate the ratio of live cells to dead cells in a certain region. The required samples include dead cell control group, live cell control group, and the sample group to be tested. Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.1. Prepare staining solution and follow the above steps to stain cells. Additionally, prepare 1 mL and 2 mL separately µ M Calcein AM and 4 µ M EthD-I solution, stain the control group according to the following instructions. For the following groups of cells or cell-free groups, it is necessary to maintain complete consistency in cell count, detection of working solution concentration, incubation time, and incubation temperature.2. Measurement of sample group and control group:A. The measured values of the sample group at 645 nm are denoted as Calcein AM and EthD-I=F (645) sam.B. The measured values of the sample group at 530 nm are denoted as Calcein AM and EthD-I=F (530) sam.C. The measurement value of dead cell EthD-I single staining control group at 645 nm is denoted as EthD-I=F (645) maxD. The measurement value of dead cell Calcein AM single staining control group at 645 nm is recorded as Calcein AM=F (645) minE. The measurement value of live cell EthD-I single staining control group at 530 nm is recorded as EthD-I=F (530) min.F. The measurement value of live cell Calcein AM single staining control group at 530 nm is denoted as Calcein AM=F (530) max.G. A blank control well without cells (with or without dye), the detection value at 530 nm is recorded as F (530) 0.H. A blank control well without cells (with or without dye), the detection value at 645 nm is recorded as F (645) 0.3. Calculate the ratio of dead cells to live cells based on measurement data:%Live Cells=(B-E) ÷ (F-E)%Dead Cells=(A-D) ÷ (C-D)Determine the ratio of live cells to dead cells in a certain areaBy creating fluorescence spectral standard curves at 530 nm and 645 nm, the number of dead and live cells can be determined, and the fluorescence intensity of each dye is linearly related to the number of dead or live cells in the sample.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. phenol red or serum may interfere with the detection of this kit. 3. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Dead and live cell staining (animal)... Read More | DescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatableDescriptionWhite LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity (sold seperately)0-30 mA variable LED outputNon-magnetic LED baseChemically resistant LED coverPTFE coated cablingPhoto Kitalysis Starter Kitrequired for operation (sold separately). Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More |