Universal Plant Total RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2113-20
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Universal Plant Total RNA Extraction Kit(SpinColumn)
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20T
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CNY470
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R2113-50
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Universal Plant Total RNA Extraction Kit(SpinColumn)
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50T
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CNY780
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R2113-200
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Universal Plant Total RNA Extraction Kit(SpinColumn)
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200T
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CNY2800
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
Lysis Buffer RL can efficiently lyse tissue cells and inactivate RNases. Combined with a specific spin column, it can extract high-purity and high-quality total RNA free from contaminants such as proteins, genomic DNA, and cellular metabolites. The extracted total RNA has good integrity, no protein or DNA contamination, and can be used in various routine molecular biology experiments, such as RT-PCR, Real-Time PCR, Northern blot, Dot blot, poly A selection, in vitro translation, cDNA library construction, and other molecular biology experiments.
Product Features:
1.The silica matrix membrane in the spin column is made of high-quality special adsorption membrane, with minimal differences in adsorption capacity between columns.
2.It combines the advantages of the guanidine isothiocyanate/phenol one-step method (good reagent stability and high purity) and the spin column (convenience and speed). No isopropanol precipitation or ethanol washing steps are required. RNA is directly eluted from the spin column, avoiding the problem of difficulty in dissolving due to excessive drying.
3.The unique formula of Lysis Buffer RL can effectively eliminate genomic DNA contamination.
4.Multiple column washing steps remove impurity proteins and cellular metabolites, resulting in high-purity extracted RNA.
Effectively reduces the content of 5S rRNA in total RNA and improves purity.
Experimental Procedures:
Pre-Experiment Preparation
1.Use sterile RNase-free centrifuge tubes, pipette tips, and other disposable plastic consumables as much as possible. If glassware or non-disposable tools are used, they should be treated with DEPC in advance or decontaminated with RNase removal products.
1) Treat the equipment and tools with 0.1% DEPC (diethyl pyrocarbonate) aqueous solution in the dark for 12 hours.
2) Then autoclave at 121°C for 30 minutes to remove residual DEPC.
2.Wear a lab coat, disposable latex gloves, and a disposable mask when preparing reagents and performing experiments to avoid artificial RNase contamination.
Experimental Procedures
1.Tissue Homogenization: Grind fresh plant tissue thoroughly in liquid nitrogen, or cut the plant tissue into pieces and grind quickly directly in Lysis Buffer RL. Add 1 mL of Lysis Buffer RL per 50-100 mg of tissue and mix well. Note: The sample volume should generally not exceed 10% of the volume of Lysis Buffer RL.
2.After vigorously vortexing the homogenized sample, let it stand at room temperature for 5 minutes to completely dissociate ribosomes.
3.Optional Step: Centrifuge at 12,000 rpm at 4°C for 10 minutes. Carefully transfer the supernatant to a new RNase-free centrifuge tube. If the sample contains a lot of impurity proteins, polysaccharides, plant tubers, nodules, etc., centrifugation can be used to remove them.
4.Add 0.2 mL of chloroform per 1 mL of Lysis Buffer RL. Tighten the tube cap, vortex vigorously for 15 seconds, and let it stand at room temperature for 3 minutes.
5.Centrifuge at 12,000 rpm in a high-speed refrigerated centrifuge at 4°C for 10-15 minutes. After centrifugation, the mixture is divided into three layers: the lower red organic phase, the middle phase, and the upper colorless aqueous phase. RNA is present in the upper aqueous phase. The volume of the aqueous phase is approximately 50-60% of the volume of Lysis Buffer RL added.
6.Carefully transfer the aqueous phase to a new RNase-free centrifuge tube (do not touch the middle phase), record the volume of the aqueous phase, add 0.5 volumes of absolute ethanol to the aqueous phase, and immediately pipette to mix well.
7.Transfer the mixed solution (less than 700 μL each time, can be added in two portions) to the Spin Column (SpinRA), centrifuge at 12,000 rpm for 45 seconds, discard the waste liquid, and reattach the spin column to the collection tube.
8.Add 500 μL of Deproteinization Buffer RE, centrifuge at 12,000 rpm at room temperature for 45 seconds, and discard the waste liquid.
9.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first), centrifuge at 12,000 rpm for 45 seconds, and discard the waste liquid.
10.Repeat step 9 once.
11.Place the Spin Column (SpinRA) back into the empty collection tube, centrifuge at 13,000 rpm at room temperature for 2 minutes to remove as much wash buffer as possible, avoiding residual ethanol in the wash buffer that may inhibit downstream reactions.
12.Remove the Spin Column (SpinRA) (avoid the bottom of the Spin Column (SpinRA) touching the waste liquid in the collection tube), place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 50~80 μL of RNase-free H₂O to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.
If more RNA is needed, the obtained solution can be re-added to the spin column and centrifuged for 1 minute, or an additional 30 μL of RNase-free water can be added and centrifuged for 1 minute, then combine the two eluates. After obtaining the RNA, store the RNA solution at -80°C to prevent degradation.
The larger the elution volume, the higher the elution efficiency. If a higher RNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30 μL. Too small a volume will reduce the RNA elution efficiency and yield.
