TRIzol Reagent目录号 : GK20008
Sample solution is provided at 25 µL, 10mM.
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Chloroform, isopropanol, 70% ethanol (DEPC water configuration), Rnase Free H2O.
Ⅰ Preparation before experiment
The key to RNA preparation is to inhibit RNA degrading enzymes in cells and prevent contamination of RNA degrading enzymes in the equipment and reagents used. Therefore, the following measures must be taken in the experiment: wear disposable clean gloves; use a special experimental bench for RNA operation; avoid talking during the operation, etc. The above methods can prevent the contamination of the experimenter's sweat and saliva by RNA degrading enzymes.
1. Try to use disposable plastic utensils. If glassware is used, it should be treated with 0.1% DEPC aqueous solution at 37°C for 12 hours before use, and then autoclaved at 120°C for 30 minutes to remove residual DEPC.
2. Reagents used for RNA experiments must be sterilized by dry heat (180°C, 60min) or in glass containers after DEPC water treatment and sterilization using the above methods (disposable plastic containers for RNA experiments can also be used) , The sterile water used must be treated with 0.1% DEPC and then autoclaved.
3. Reagents and sterile water for RNA experiments should be dedicated to avoid cross-contamination after mixing.
Ⅱ Experimental operation
The usage of TRIzol Reagent is as follows
|Sample type||Sample size||Usage of TRIzol|
|Adherent cultured cells||10 cm2||1 mL|
|Suspension culture cells||1x106-10x106||1 mL|
|Common tissue samples (muscle, etc.)||50-100 mg||1 mL|
|Special tissue samples (liver, spleen, etc.)||30-50 mg||1-2 mL|
|Plant tissue||30-50 mg||1 mL|
Sample size and RNA yield
|Sample type||Sample size||RNA yield|
|Plant tissue||25 mg||10~20 μg|
|Tissues such as muscle/brain||50 mg||10~25 μg|
|Liver||50 mg||100~300 μg|
TRIzol usage instructions
|Adherent cells||Suspension cells, yeast, bacteria||Animal and Plant tissue|
|1. Sample pretreatment||Pour out the culture solution from every 10 cm2 of the cultured cells and wash them with PBS once to remove as much excess solution as possible.||Pour the suspension cultured cells together with the culture solution into a centrifuge tube, centrifuge at 8,000 rpm for 2 min, discard the supernatant, and add 50μl of sterile water to resuspend the cells until there is no obvious precipitation.||Transfer the sample to a mortar pre-cooled with liquid nitrogen, grind the tissue with a pestle, and continuously add liquid nitrogen in the meantime until it is ground into a powder.|
|2. Add TRIzol||Add 1 ml of TRIzol to distribute the lysate evenly on the cell surface, and then use a pipette to blow the cells off. Transfer the cell lysate to a 1.5ml EP tube.||Add 1ml TRIzol.||Add the ground tissue to a 1.5 ml EP tube containing 1 ml TRIzol.|
|3. Lysed sample||After adding TRIzol, immediately turn it upside down with wrist force until the cells and tissue powder are evenly dispersed without lumps. Leave it at room temperature for 5 minutes to completely separate the nucleic acid-protein complexes.|
|4. Add Chloroform||Add 200 μl chloroform, shake vigorously with the wrist for 15 seconds, and leave it at room temperature for 2 minutes.|
|5. Centrifugal layering||Centrifuge at 13,000 rpm for 10 minutes, and transfer 600 μl of colorless supernatant to a new 1.5EP tube.|
|6. Add isopropanol||Add 600 μl of isopropanol to the above 600 μl of supernatant, turn it upside down several times vigorously with the wrist, and place it at -20°C for 5 minutes.|
|7. Centrifugation of total RNA||Centrifuge at 13,000 rpm for 10 min, carefully discard the supernatant, and save the bottom total RNA pellet.|
|8. Rinse total RNA||Add 1ml of 70% ethanol to each tube of the pellet, turn it upside down several times, and centrifuge at 13,000 rpm for 5 min. Carefully discard the supernatant and save the bottom RNA pellet.|
|9. Repeat the rinse one more time||Repeat step 8 and wash again.|
|10. Volatile residual ethanol||Pour off the washing solution, centrifuge again for a short time for 10 seconds, absorb the remaining washing solution with a 10 μl tip, and place it at room temperature to evaporate the ethanol (~20min).|
|11. Dissolve total RNA||Add 20-100μl TE Buffer or RNase Free H2O to each tube to dissolve total RNA.|
1. Low extraction rate. Possible reasons: (a. Sample lysis or homogenization is not complete; b. RNA precipitation is not completely dissolved)
2.A260/A280<1.65. Possible reasons: (a. When measuring the absorbance, the RNA sample was not dissolved in water, but dissolved in TE; b. The amount of tissue added when the sample was homogenized was too much; c. After stratification, the supernatant was less than 500μl; d. The organic phase was mixed in the water phase)
3. Excessive DNA contamination. Possible reasons: (a. The amount of reagents added during sample homogenization is too small or the amount of tissue is too much; b. The sample contains organic solvents)
TRIzol Reagent is a product that can extract RNA from animal and plant samples. The sample can be fully lysed in TRIzol Reagent. During the homogenization or lysis of the sample, it can maintain the integrity of the RNA, while lysing the cells and dissolving the cell contents. TRIzol Reagent has a strong broad spectrum and can be applied to the extraction of total RNA from various samples. The extraction process is convenient and fast, and the entire operation can be completed within one hour. This reagent can be used for small samples (50-100 mg tissue, 1x106 cells) and also suitable for large samples (≥1g tissue, >107 cells). It is applicable to humans, animals, plant tissues, and bacteria, and can process a large number of different samples at the same time, and the entire extraction process can be completed within one hour. The isolated total RNA protein and DNA are extremely low in contamination, and can be used for Northern Blot, reverse transcription, polyA screening, in vitro translation, RNase protection analysis, and gene cloning.
TRIzol试剂是一种可以从动物和植物样本中提取RNA的产品。在TRIzol试剂的作用下，样本可以完全裂解。在样品均质或裂解过程中，它能够保持RNA的完整性，同时溶解细胞并溶解细胞内容物。 TRIzol试剂具有强大的广谱性，并可应用于从各种样品中提取总RNA。提取过程方便快捷，整个操作可以在一个小时内完成。该试剂可用于小样本（50-100毫克组织、1x106个细胞），也适用于大样本（≥1克组织、＞107个细胞）。它适用于人类、动物、植物组织和细菌，并且可以同时处理大量不同类型的样品，在一个小时内完成整个提取过程。分离出来的总RNA蛋白质和DNA极低污染，并可用于Northern Blot、反转录、polyA筛选、体外翻译、RNase保护分析和基因克隆等实验研究。
|应用&特点||• Permits the isolation of RNA, DNA, and protein from the same sample
• Offers superior lysis capability, even with difficult sample types
• Optimized formulations and protocols for tissues, cells, serum, virus, and bacteria
|运输方式||Ship with blue ice.|
|储存条件||Store at 2-8°C protected from light for 2 years.|