StarPure Plus Complex Plant total RNA Extraction Kit It is suitable for rapid extraction of total RNA from plant tissues, particularly those rich in polysaccharides, polyphenols, or starch (such as cotton, mature rice, pine needles, poplar, loquat, potato, banana, grape, apple, pear, rose, buckwheat seeds, and Arabidopsis seeds). A unique lysis buffer rapidly lyses cells and inactivates cellular RNases. Polysaccharides, polyphenols, and secondary metabolites are removed by centrifugation. The lysis mixture is then ethanol-adjusted for RNA binding and adsorption to a genomic DNA cleanup column. RNA is then eluted through selective elution, while DNA remains on the column and is discarded. After the washed RNA is ethanol-adjusted for binding, it selectively adsorbs to the silica matrix membrane within the spin column under a high-salt state. A series of rinses and centrifugation steps remove impurities such as cellular metabolites and proteins. Finally, a low-salt RNase-free H 2 Purified RNA is eluted from the silica-based membrane. The resulting RNA generally does not require DNase digestion and can be used in experiments such as reverse transcription PCR and fluorescent quantitative PCR.
StarPure Plus Complex Plant total RNA Extraction Kit
StarPure Plus Polysaccharide and Polyphenol/Complex Plant Sample Total RNA Extraction Kit
Product Advantages
Product Application This kit is suitable for rapid extraction of total RNA from plant tissues, especially those rich in polysaccharides, polyphenols or starch. The obtained RNA can be used for reverse transcription PCR, fluorescence quantitative PCR and other experiments. Product components  Experimental procedures Transfer 1 mL of Lysis Buffer (CLB) to a centrifuge tube (if CLB has precipitated, re-dissolve it in a 65°C water bath). Add 5% β-mercaptoethanol to the lysate (50 μL β-mercaptoethanol per 1 mL of CLB). Invert to mix thoroughly and preheat in a 65°C water bath. Scale up the sample for multiple samples. 1. Direct grinding method (recommended when there is no liquid nitrogen in the laboratory or when the plant samples are soft and easy to grind): a. Weigh 100-200 mg of fresh or frozen plant samples (100-150 mg for low-water samples such as leaves and seeds, and more for high-water samples such as strawberries and watermelons). Quickly cut into small pieces and place in a mortar. Add 1 mL of CLB (prepared with β-mercaptoethanol) and grind thoroughly at room temperature to form a homogenate. Grind quickly to allow immediate and full contact between the tissue and the CLB lysis buffer to inhibit RNase activity.✮Note: β-mercaptoethanol is a lysis buffer CLB The final concentration can be increased to 10-20% when necessary. If the plant is particularly complex, you can try adding PVP 40 to a final concentration of 2%. b. Transfer the lysate to a centrifuge tube and immediately shake vigorously for 15 seconds. Return the tube to a 65°C water bath for 5-10 minutes, inverting 1-2 times to mix thoroughly. Centrifuge at 13,000 rpm for 10 minutes to pellet any insoluble fragments.c. Transfer the lysate supernatant (taking as much as possible without exceeding the capacity of the gDNA cleanup column) to a fresh centrifuge tube. Add 5 times the volume of anhydrous ethanol as the supernatant and immediately mix by pipetting. Do not centrifuge. If there are floating objects on the surface of the supernatant, use a pipette to pick them up and absorb the liquid below. d. Immediately proceed to step 3 of the procedure.2. Liquid nitrogen grinding method (This method is widely applicable and is recommended for extracting complex, difficult to break, and easily degradable samples) : a. Grind fresh or -70°C frozen material in liquid nitrogen to a fine powder.b. Transfer 100-200 mg of the fine powder (100-150 mg for low-water samples such as leaves and seeds, and more for high-water samples such as strawberries and watermelons) to a preheated centrifuge tube containing lysis buffer CLB (pre-added with β-mercaptoethanol). Immediately vortex vigorously for 30-60 seconds or pipette until a satisfactory homogenate is obtained (or perform an electric homogenization for 30 seconds). This will shear the DNA, reduce viscosity, and increase yield. c. Immediately return the tube to a 65°C water bath for 5-10 minutes, inverting 1-2 times to mix thoroughly. d. Centrifuge at 13,000 rpm for 10 minutes to pellet the insoluble fragments. e. Transfer the supernatant (taking as much as possible without exceeding the capacity of the gDNA cleanup column) to a fresh centrifuge tube. Add 5 times the volume of anhydrous ethanol as the supernatant and immediately mix by pipetting. Do not centrifuge. If there is any floating material on the surface of the supernatant, remove it with a pipette tip and aspirate the liquid below. f. Immediately proceed to step 3 of the operating instructions. 3. Transfer the mixture (less than 720 μL each time, which can be added in two batches) to a gDNA cleanup column (coated with a collection tube), centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid. ✮ Note: Ensure that all liquid is filtered through after centrifugation and no residue remains on the membrane. To increase the centrifugal force and centrifugation time. 4. Place the gDNA cleanup column in a clean 2 mL centrifuge tube (RNase-free or DEPC-treated is not necessary; a clean new centrifuge tube will generally suffice. Alternatively, use a new, clean collection tube that comes with the RNA adsorption column). Add 500 μL of RLT Lysis Buffer to the gDNA cleanup column and centrifuge at 13,000 rpm for 30 seconds. Collect the filtrate (RNA is in the filtrate). Accurately estimate the filtrate volume (usually around 450-500 μL, minus the volume lost during filtration). Add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this point, but this will not affect the extraction process. Immediately mix by pipetting without centrifuging. 5. Immediately add the mixture (less than 720 μL each time, which can be added in two batches) to an adsorption column (place the adsorption column in a collection tube) and centrifuge at 13,000 rpm for 2 minutes. Discard the waste liquid. ✮ Note: Make sure that all the liquid is filtered through after centrifugation and there is no residue on the membrane. If necessary, increase the centrifugal force and centrifugation time. 6. Add 700 μL of Protein Wash Buffer, incubate at room temperature for 1 minute, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid. 7. Add 500 μL Wash Buffer (please check to see if anhydrous ethanol has been added beforehand!), centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid. Repeat the wash cycle. 8. Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much of the rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions. 9. Remove the RA adsorption column and place it in an RNase-free centrifuge tube. Depending on the expected RNA yield, add 30-50 μL of RNase-free water (preheated in a 70-90°C water bath) to the middle of the adsorption membrane. Incubate at room temperature for 1 minute and centrifuge at 12,000 rpm for 1 minute. 10. If the expected RNA yield is >30 μg, add 30-50 μL of RNase-free water and repeat step 9. Combine the two washes, or use the first eluate and add it back to the adsorption column and repeat the step (if a higher RNA concentration is required). ✮ Note: Eluted twice RNA High eluent concentration , Elution was performed twice and the eluates were combined. RNA The yield is 15–30% higher than the former , But the concentration should be low , Users choose according to their needs.
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