Products and features Construction of cDNA libraries, in vitro translation of proteins, and detection of rare mRNA by RT-PCR methods often require the isolation of mRNA from total RNA. The mRNA only accounts for about 1% of the total RNA. Therefore, it can be separated from other RNA (such as rRNA, tRNA, etc.) by affinity binding with Oligo (dT) cellulose. This product is specially used for one-stop extraction of mRNA from plant tissues. It has the following characteristics: 1. One-stop, it is a combination of high-efficiency, polysaccharide-free column plant RNA extraction reagents and mRNA extraction reagents with Tianze gene. It is ready to use, very convenient. 2. It can process 50-500 mg of plant tissue at a time, and can obtain about 10-15ug of total RNA, from which about 0.1-0.5ug of mRNA can be obtained. 3. Oligo (dT) cellulose has strong adsorption capacity, and can adsorb 50-80 OD of mRNA per gram, which is equivalent to 2000-3000ug of mRNA. 4. The operation process of mRNA is simple, and it can be completed only by centrifugation, eliminating the cumbersome operations such as conventional column filling and column washing. 5. The obtained mRNA can be directly used to construct cDNA library, in vitro translation of proteins and RT-PCR. 25 times packaging Column plant RNAout solution A 50 mL Column plant RNAout solution B 15 mL Column plant RNAout solution C 50 mL 50 sets of centrifugal adsorption columns Universal column wash 50 mL RNA eluent 10 mL Binding liquid 100mL Oligo (dT) cellulose 25mg RNase-free water 10mL Trace nucleic acid precipitation agent 10mL 1 manual Transportation and storage Normal temperature transportation, storage at 4 ℃, Oligo (dT) cellulose low temperature transportation, storage at -20 ℃, valid for one year. Bring your own reagent chloroform, RNase-free collection tube. The first step of the method is to extract total plant RNA using plant RNAout, and finally elute with 50uL, collect the two tubes together to obtain a total of 100uL total RNA (see below for details): 1. Estimate the amount of plant tissue cells. Each micro-extraction generally requires 100-200 mg plant leaves, or 50-100 mg plant seeds, or 200-500 mg plant fruits. 2. First cut the fresh plant tissue into small pieces (the plant tissue stored in RNALOCKER needs to absorb the RNALOCKER liquid with paper and then cut into small pieces), put into a 10-15 mL plastic centrifuge tube, add 1 mL Solution A is then homogenized with a homogenizer for 5-20 seconds. Foam will be generated during homogenization, but it will not affect the extraction effect. You can also use the liquid nitrogen inkstone grinding method to break up the plant tissue, and add 1 mL of solution A after the inkstone grinding. Note: Dissolution solution A precipitates. Solution A may produce a precipitate after being placed at 4 ° C. Before use, it must be placed in a 65 ° C water bath to completely dissolve the precipitate and shake it thoroughly before use. 3. Transfer the homogenate or inkstone to a clean 1.5 mL plastic centrifuge tube (you may not need to transfer non-liquid cell debris). Some plant tissues (such as fruits) contain a lot of water, the homogenate will be more than 1 mL, and only 1 mL is taken during transfer. 4. Add 0.3 mL of solution B and 0.2 mL of self-prepared chloroform to the centrifuge tube, shake on a shaker for 30 seconds and mix well. At this time, the solution is uniformly milky. When the oscillator oscillates, the solution at the bottom of the tube must be shaken. 5. Centrifuge at 12000 rpm at room temperature for 3-5 minutes. There will be about 5-10 mm thick cell disruption between the two phases. 6. Transfer the supernatant (approximately 0.6 mL) to another clean 1.5 mL plastic centrifuge tube. The lower organic phase and the middle layer contain DNA, protein, and other impurities to avoid touching or aspiration. It is best to leave 100 uL of supernatant untaken. 7. Add an equal volume of solution C and mix thoroughly by inverting. 8. Transfer half of the solution to a centrifuge adsorption column (60911B), centrifuge at 12000 rpm at room temperature for half a minute, and discard the penetrant. 9. Transfer the remaining half of the solution to the same centrifugal adsorption column, centrifuge at 12000 rpm at room temperature for half a minute, and discard the penetrant. 10. Add 0.7 mL of universal column washing solution, centrifuge at room temperature for half a minute, and discard the penetrating solution. 11. Add 0.3 mL of universal column washing solution, centrifuge at room temperature for half a minute, and discard the penetrating solution. This step can be omitted. 12. Centrifuge at room temperature for half a minute. This step is very important, otherwise the residual ethanol will affect the use of RNA. 13. Transfer the centrifugal adsorption column to the RNase-free collection tube, add 50-100 uL RNA eluent, and leave at room temperature for 1-2 minutes. 14. Centrifuge at 12000 rpm at room temperature for half a minute. The solution in the centrifuge tube is the RNA sample, which can be used immediately or stored at -80 ° C until use. The second step is to purify mRNA from total RNA (see below for details): This product provides 25 mg of Oligo (dT) cellulose. Each gram of Oligo (dT) cellulose can bind 50-80 OD of mRNA. 1. Weigh 1 mg of Oligo (dT) cellulose in a 1.5 mL plastic centrifuge tube. Add 0.5 mL of the binding solution to a centrifuge tube containing 1 mg Oligo (dT) cellulose, mix well, and leave at room temperature for 5 minutes. You can also add 250 uL of the binding solution to a centrifuge tube containing 25 mg Oligo (dT) cellulose, take 10 uL each time, equivalent to 1 mg of Oligo (dT) cellulose, and mix with 0.5 mL of the binding solution, at room temperature Leave for 5 minutes. Store the remaining part at -80 ℃. 2. Centrifuge at 200-1000 g for 30 seconds, carefully aspirate the supernatant and leave Oligo (dT) cellulose precipitate for use. 3. Heat 100 uL of the total RNA prepared in the first step (if the volume is less than 100 uL, make up with water), and heat at 65 ° C for 5 minutes. 4. Add equal volume of binding solution, mix and cool to room temperature, then transfer to a centrifuge tube containing Oligo (dT) cellulose. 5. After mixing, let stand at room temperature for 5 minutes. Invert during mixing several times. 6. Centrifuge at 200-1000 g for 5 minutes at 4 ° C. Carefully transfer the supernatant to a centrifuge tube. This supernatant is total RNA from which mRNA has been removed. It can be stored in the refrigerator as a negative control. 7. Add 0.5 mL of binding solution, mix and centrifuge at 200-1000 g at 4 ° C for 5 minutes. Discard the supernatant carefully. 8. Repeat the previous step 2-4 times. 9. Add 50 uL of RNA eluent, mix and centrifuge at 200-1000 g at 4 ° C for 5 minutes, carefully collect the supernatant (mRNA). 10. Repeat the previous step 1-3 times. 11. The mRNA collected each time is mixed, which is the mRNA solution. This solution can be used directly. If it is too thin, it can be concentrated according to the third step. The third step is to precipitate mRNA with a trace amount of nucleic acid precipitant (see below for detailed operation): 1. Mix this product with the mRNA solution obtained in the third step at a ratio of 2: 1. Note: The trace amount of nucleic acid precipitant has insoluble nucleic acid precipitant, so it needs to be shaken before use so that the part taken contains a certain amount of nucleic acid precipitant. 2. Centrifuge at 15,000 g for 10 minutes at room temperature (if the nucleic acid content is below 2 ng, it is recommended to centrifuge for 30 minutes). 3. After discarding the supernatant, add 1 mL of 75% ethanol and mix by shaking. 4. Centrifuge at 15000 for 3-5 minutes and discard the supernatant. 5. After briefly centrifuging, carefully aspirate the supernatant. The precipitate is the recovered mRNA precipitate, which can be used directly in DEPC water or stored at -70 ℃ for a long time. Makeup Brushes,Wooden Handle Makeup Brushes,Bling Makeup Brushes,5Pcs Brush Set DONGGUAN YACAI COSMETICS CO.,LTD. , https://www.yacaicosmetic.com