| Description | Inquire | Inquire | Format:2-ComponentEnzyme:Horseradish peroxidase | Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid preparation of genomic DNA and PCR amplification, and is suitable for one-step extraction of genomic DNA from various plant and animal tissues and bacteria and for PCR amplification. The whole extraction process does not require liquid nitrogen grinding, organic solvent extraction, anhydrous ethanol precipitation, and the quality of extracted DNA is stable. The 2×PCR MasterMix provided in this kit is a highly compatible PCR reagent that can amplify DNA samples efficiently and specifically, which includes DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and so on. It is characterized by fast and easy, high sensitivity, high specificity, good stability, etc. It is especially suitable for high throughput screening.Pre-experiment Preparation and Important Notes1. Add the specified amount of 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 samples should be avoided, as this will result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Before use, please check Buffer SA for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer SA in a 56℃ water bath.4. The PCR MasterMix provided with this product is 2×, when using it, you need to add template and primer, and add RNase-Free Water to make up the volume, so that its concentration is 1× to carry out the reaction.Procedure1. Fetch:Plant material: take about 10 mg of sample in a centrifuge tube (provided); Animal material: take about 10 mg of sample in a centrifuge tube (provided);Bacteria: Take 200-800 µL of bacteria in good growth condition in a centrifuge tube (self-provided) and collect the bacteria.2. Add 200 µL of Buffer SA and vortex to mix.Note: In the case of plant leaves and animal tissues, they should be ground with a pestle and mortar as much as possible: in the case of plant seeds, they should be crushed and finely ground beforehand; bacterial and 1-3 mm rat-tail samples can be directly vortex lysed.3. Add 10µL of Proteinase K, mix well, incubate at 56℃ for 10 minutes, and treat at 95℃ for 5 minutes.Note: 1) In the case of animal tissue samples, the incubation time at 56°C may be extended to 30 minutes as appropriate; if there is any incompletely digested tissue, it should be removed as thoroughly as possible after centrifugation in the next step.2) Be careful not to exceed 5 minutes when treating at 95°C.4. 13,000 rpm (~17,900 x g), centrifugation for 5 minutes.5. Transfer the supernatant to a new centrifuge tube (self-prepared) and use it directly for PCR amplification, or store the solution at 4℃ or -20℃.6. PCR amplification:1) PCR reaction system:The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.reagents20 µL systemfinal concentration2×PCR MasterMix10 µL1×Forward Primer, 10 µM1 µL0.4 µMReverse Primer, 10 µM1 µL0.4 µMTemplate DNA1-2 µL RNase-free Waterup to 20 µLNote: Please use the final concentration of 0.2-0.6µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the concentration of primer can be increased; if a non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.2）PCR reaction conditions:movetemptimingpremutability94°C2mindenaturation94°C30sannealing (metallurgy)55-65°C30s30-40 cyclesreach72°C60sultimate extension72°C5minNote: 1) In general, the annealing temperature is 5℃ lower than the melting temperature of the amplification primer Tm, and the annealing time is generally 30-60 seconds. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when a non-specific reaction occurs, the annealing temperature should be raised, thus optimizing the reaction conditions.(2) The extension time is set according to the size of the fragment to be amplified, and the amplification efficiency of Taq DNA Polymerase included in this product is 1kb/30s. 3) The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.(3) Result detection: 5 µL of reaction product was taken at the end of the reaction and directly detected by agarose gel electrophoresis... Read More | O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water 10 mL RT O665690G Spin Columns FS with Collection Tubes 50 EA RT O665690H Spin Columns RM with Collection Tubes 50 EA RT O665690I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProduct IntroductionThis kit is suitable for extracting RNA from a wide range of plants, even from plants rich in polysaccharides and polyphenols, high quality RNA can be successfully extracted, such as rice leaves, wheat leaves, corn leaves, tobacco leaves, pine needles, ginkgo leaves, poplar leaves, pomegranate leaves, holly leaves, apples, peaches, pears, tomatoes, cherries, apricots, bananas, grapes, loquats, cinnamon rinds, cinnamon pulp, lychee fruit rinds, lychee pulp, soybean, peanut, corn, potato tuber, moonflower petal, pomegranate petal, shiitake mushroom, flat mushroom and other samples. The unique lysate formula can rapidly inactivate the RNA enzyme in the cell, effectively remove the effect of polysaccharide and polyphenol on RNA extraction, without the need for phenol, chloroform and other reagents, while using silicon matrix membrane adsorption of RNA for purification, the total RNA extracted is highly pure, without the contamination of genomes, proteins and other impurities, and can be used for Real Time RT-PCR, RT-PCR, It can be used for Real Time RT-PCR, RT-PCR, Northern Blot, Dot Blot, in vitro translation and other downstream experiments.RNA yieldSelf-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction)Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips.(2) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the rate and quality of RNA extraction.3. If Buffer RLS produces a precipitate, heat to dissolve it and leave at room temperature.4. Please add β-mercaptoethanol to Buffer RLS before use, add 20µl β-mercaptoethanol to 1ml Buffer RLS. Buffer RLS with β-mercaptoethanol can be stored for 1 month at room temperature.5. Anhydrous ethanol should be added according to the instructions on the reagent bottle label before using Buffer RW2 for the first time. Operation steps1. Homogenization: Take 50-100mg of plant tissue and quickly grind it into powder in liquid nitrogen, add 500µl of Buffer RLS (please check whether β-mercaptoethanol is added before use), and immediately mix it by vortexing with vigorous shaking.Note: For materials that are extremely rich in water content, such as watermelon pulp, tomato, pear pulp, etc., more material can be added appropriately, up to 200 mg; for starch-rich samples or mature leaves, the amount of Buffer RLS can be increased appropriately, up to 700 µl.2. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min at 4°C.3. Transfer the supernatant into the filter columns (Spin Columns FS) that have been loaded into the collection tubes, centrifuge at 12,000 rpm at 4°C for 1 minute, carefully aspirate the supernatant in the collection tubes and transfer it to new RNase-Free centrifugation tubes (self-provided), avoiding the tip of the gun from touching the cell debris precipitation in the collection tubes as much as possible.4. Slowly add 0.5 times the volume of the supernatant in anhydrous ethanol, mix well (a precipitate may appear), and transfer the resulting solution together with the precipitate to a Spin Columns RM in a collection tube, or in two batches if you cannot add all of the solution at once. centrifuge the column for 1 minute at 12,000 rpm at 4°C. Dispose of the spent solution and place the column back into the collection tube. Centrifuge at 12,000 rpm for 1 minute at 4°C, discard the spent solution and return the column to the collection tube.5. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and prepare a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.9. Add 500 µl of Buffer RW2 to the adsorbent column RM (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute at 4°C, discard the waste solution and put the adsorbent column back into the collection tube.10. Repeat step 9.11. Centrifuge at 12,000 rpm for 2 minutes at 4°C.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 ml), add 30-50 µl of RNase-Free Water dropwise to the middle part of the adsorption membrane overhang, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 1 min, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... Read More |