Transportation temperature: normal temperature transportation

Product Introduction:

This reagent is a universal total RNA extraction reagent suitable for the extraction of total RNA from animal and plant cells or tissues, as well as bacteria. It can effectively prevent the degradation of RNA during the extraction process. The obtained RNA can be directly used for a series of operations such as Northern hybridization, mRNA purification, in vitro translation, RNase protection experiments, RT-PCR, and cDNA cloning. This reagent can be used for 100 total RNA extractions (10 square centimeter cells or 100mg tissue).

Operating instructions:

Bring your own newly opened or specialized chloroform, isopropanol, 75% ethanol, and DEPC treated water.

1. Cell lysis or tissue homogenization:

1) Adhering cells: Suck up the culture medium, add 1 ml TriQuick Reagent to every 10 square centimeters (6-well plate or 35 mm plate) of cells to cover the cultured cells, and then blow 2-3 times with a pipette or sampler. The cells should be completely lysed, and then transfer to a centrifuge tube.

2) Suspension cells: Collect cells by centrifugation, aspirate the liquid, and add 1ml TriQuick Reagent for every 5 to 10 million (5 × 106-1 × 107) animal, plant, or yeast cells, or 10 million (1 × 107) bacteria. Blow it with a straw or sampler to completely lyse it. If certain yeast and bacteria are not fully lysed, they can be homogenized using a homogenizer to ensure complete lysis. Transfer to centrifuge tube.

3) Organization: Cut the tissue into small pieces and place them in a glass homogenizer. Frozen tissues can be ground into homogenates in a mortar. Add 1ml TriQuick Reagent to every 50-100mg tissue and homogenize until completely lysed. Transfer to centrifuge tube.

The cracking product should be a clear, transparent, viscous liquid. Leave at room temperature for 5 minutes. For tissue samples rich in impurities such as polysaccharides and proteins, insoluble substances may still remain after homogenization. They can be centrifuged at 4 ℃ for 10 minutes at 12000g, and then the supernatant can be transferred to a new centrifuge tube.

2. Separation: Add 0.2 times the volume of chloroform (1 ml TriQuick Reagent to 0.2 ml chloroform) into a centrifuge tube containing the lysate, shake well on an shaker for 30 seconds, and let it stand at room temperature for 2-3 minutes. Centrifuge at 4 ℃ for 10 minutes at 12000g, then transfer the upper aqueous phase containing total RNA into a new centrifuge tube. Each milliliter of TriQuick Reagent can absorb approximately 0.5-0.6 ml. The organic phase and intermediate layer contain DNA and proteins, and should be avoided from contact.

3. Precipitation: Add 0.5 ml of isopropanol per milliliter of TriQuick Reagent, invert and mix several times, and precipitate at room temperature for 10 minutes. Centrifuge at 4 ℃ for 10 minutes at 12000g, and RNA precipitation can be seen at the bottom of the tube. Discard the supernatant and add 1 ml of 75% ethanol per milliliter of TriQuick Reagent. Gently invert and mix well to clean the RNA precipitate. Centrifuge 12000g at 4 ℃ for 2 minutes, discard the liquid, be careful not to discard the RNA precipitate. Invert and dry at room temperature for 5-10 minutes.

4. Dissolution: Add an appropriate amount of DEPC treated water to dissolve the RNA precipitate. Store at -80 ℃.

matters needing attention:

1. The vessels, centrifuge tubes, and tips used in the RNA extraction process should be free of contamination and RNA enzymes. Caution should be exercised during operation to prevent contamination of the sample by exogenous RNA enzymes, which may lead to degradation of the RNA sample. 2. The reagent contains harmful substances such as phenols, please pay attention to personal protection.