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Trizol Pal nucleic acid extractor replaces chloroform reagent

NegotiableUpdate on 05/06

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Nature of the Manufacturer: Producers

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Overview

Trizol Pal nucleic acid extractor replaces chloroform reagent for RNA extraction. Trizol pal (chloroform replacement reagent) can be used in combination with Trizol Reagent (item number: R1000) to replace chloroform and repeatedly extract proteins from lysis systems. It has a wide range of applications and can extract total RNA from animal tissues, plant materials, various microorganisms, and cultured cells. The sample is fully lysed in Trizol while maximizing the integrity of RNA.

Product Details

Trizol Pal Chloroform Reagent Used for RNA ExtractionItem number: R1200

Storage conditions: Store in the dark at 0-30 ℃

Trizol Pal Chloroform Reagent Used for RNA ExtractionProduct Description:

Trizol PAL can be used in conjunction with Trizol Reagent (item number: R1000) to replace chloroform for repeated extraction of lysis systems to remove proteins. It has a wide range of applications and can extract total RNA from animal tissues, plant materials, various microorganisms, and cultured cells. The sample is fully lysed in Trizol while maximizing the integrity of RNA. After centrifugation with Trizol PAL, the solution will be divided into three layers: the upper colorless aqueous phase, the middle layer, and the lower red organic phase, with RNA distributed in the supernatant layer. After collecting the supernatant, total RNA can be recovered by precipitation with isopropanol. The extracted total RNA has good integrity, no protein or DNA contamination, and can be used for various routine molecular biology experiments, such as RT-PCR、Real-time RT-PCR、Northern Blot、Dot Blot、 External translation, etc.

Self provided reagents

Trizol Reagent (item number: R1000), isopropanol, 75% ethanol, RNase free water (newly opened or specifically for RNA extraction)

Nucleic Acid Extraction InstrumentNotes:

1. To prevent RNase contamination, attention should be paid to the following aspects:

1) Use RNase free plastic products and gun tips to avoid cross contamination.

2) Glassware should be dry baked at 180 ℃ for 4 hours before use, while plastic utensils can be soaked in 0.5 M NaOH for 10 minutes, rinsed with water, and sterilized under high pressure.

3) The solution should be prepared using water without RNase.

4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.

2. The extracted samples should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction.

3. When using this product, protective equipment such as gloves, eye masks, face shields, etc. should be worn. If accidentally in contact with the eyes, rinse immediately with plenty of water and seek medical treatment. Keep away from sources of fire and heat when using.

3. RNA precipitation stored in 75% ethanol can be stored for one week at 2-8 ℃ and for one year at -20 ℃.

4. RNA has a relatively short half-life and is easily degraded. It is recommended to conduct subsequent experiments as soon as possible after extraction, such as reverse transcription into cDNA, Northern Blot, etc.

5. If downstream experiments are highly sensitive to DNA, it is recommended to use DNase I without RNase to treat RNA.

Operation steps:

1. Handling of various materials

1.1 Plant tissue: Take fresh plant tissue and grind it thoroughly in liquid nitrogen or cut the plant tissue into small pieces and quickly grind it in Trizol. Add 1mL Trizol to every 30-50mg of tissue and mix well.

Note: The sample volume should generally not exceed 10% of the Trizol volume.

1.2 Animal tissue: Fresh or -70 ℃ frozen animal tissue should be cut into small pieces as much as possible, and 1 mL of Trizol should be added to every 30-50 mg of tissue for homogenization treatment using a homogenizer. Or grind in liquid nitrogen and add 1 mL of Trizol to mix well.

Note: The sample volume should generally not exceed 10% of the Trizol volume.

1.3 Single layer cultured cells: Remove the culture medium and directly add an appropriate amount of Trizol (1 mL Trizol is required per 10 cm2 area) to the culture plate. Use a sampler to repeatedly blow and beat to lyse the cells. Alternatively, after treatment with trypsin, the cell solution can be transferred to a RNase Free centrifuge tube and centrifuged at 300 × g for 5 minutes. Collect the cell pellet, carefully remove all supernatant, and add 1 mL of Trizol to mix well.

Note: 1) The number of collected cells should not exceed 1 × 10 ^ 7.

2) The dosage of TRNzol is determined by the area of the culture plate, not by the number of cells. If the dosage of Trizol is insufficient, it may lead to DNA contamination in the extracted RNA.

3) When collecting cells, it is necessary to remove the cell culture medium completely, otherwise it will lead to incomplete lysis and a decrease in RNA production.

1.4 Cell suspension: Collect cells by centrifugation. Add 1 mL of Trizol every 5 × 10 6-1 × 10 7 animal, plant, and yeast cells, or every 10 7 bacterial cells.

Note: 1) Do not wash cells before adding Trizol to prevent RNA degradation.

2) Some yeast and bacterial cells may require homogenization or liquid nitrogen grinding treatment.

1.5 Blood processing: Take fresh blood directly and add 3 times the volume of Trizol (recommended to add 0.75 mL Trizol to 0.25 mL whole blood), shake thoroughly and mix well.

1.6 Optional steps: For samples with high protein, fat, polysaccharide, or extracellular substance content, such as muscle tissue, adipose tissue, or plant tubers, centrifugation at 12000 rpm (~13400 × g) for 10 minutes after homogenization treatment can be performed to remove insoluble substances. At this time, the precipitate contains extracellular substances, polysaccharides, and high molecular weight DNA, while RNA is present in the supernatant.

2. After adding Trizol to the sample, repeatedly blow and beat it several times to fully lyse the sample. Let it stand at room temperature for 5 minutes to completely separate the protein nucleic acid complex.

3. Add Trizol Pal to the above solution, with 0.2 mL of Trizol Pal added for every 1 mL of Trizol used. Cover the tube and shake vigorously for 15 seconds, then let it sit at room temperature for 2-3 minutes.

4. Centrifuge at 12000 rpm at 4 ℃ for 15 minutes, and the sample is divided into three layers: a red organic phase, a middle layer, and an upper layer of colorless aqueous phase. RNA is mainly in the aqueous phase. Transfer the aqueous phase (about 600 μ L) to a new RNase Free centrifuge tube (provided).

5. Add an equal volume of isopropanol to the obtained aqueous solution, invert and mix well, and let it stand at room temperature for 10 minutes.

6. Centrifuge at 12000 rpm at 4 ℃ for 10 minutes and discard the supernatant.

7. Wash the precipitate with 75% ethanol (prepared with RNase free water). Wash the precipitate with 1 mL of 75% ethanol for every 1 mL of Trizol used.

8. Centrifuge at 12000 rpm at 4 ℃ for 3 minutes, be careful not to aspirate the supernatant, and be careful not to aspirate the RNA precipitate.

Note: The remaining small amount of liquid can be briefly centrifuged and then sucked out with a gun tip, being careful not to discard the sediment.

9. Leave at room temperature for 2-3 minutes and air dry. Add 30-100 μ L of RNase free water, dissolve RNA thoroughly, and store the obtained RNA at -70 ℃ to prevent degradation.

Note: The precipitate should not be excessively dried to avoid difficulty in dissolution.