Reagent for simultaneous extraction of DNA and RNA, extraction method and application
A technology of reagents and organic solvents, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of difficulty in extracting high-quality RNA, cumbersome operation steps, no reagents, etc., and achieve simple and fast operation methods with high purity and the effect of high concentration and low sample requirement
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Embodiment 1
[0072] (1) Processing of tissue samples
[0073] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 1 of Table 1 was added, shaken at 200 rpm for 5 minutes at room temperature, centrifuged at 12000 g for 15 minutes at room temperature, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction. The density of pure phenol is 1.07g / cm 3 , which is located in the lower layer of the aqueous phase when mixed with water. Chloroform makes the separation of the two solvents more obvious because chloroform is miscible with phenol and has a greater density of 1.47 g / cm 3 .
[0074] (2) RNA extraction:
[0075] (1) Carefully take out the upper layer liquid, add isopropanol drop by drop, shake slowly at room temperature 100rpm for 10 minutes, centrifuge at room temperature 12000g for 15 minutes, discard all the ...
Embodiment 2
[0085] (1) Processing of tissue samples
[0086] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 2 of Table 1 was added, shaken at 100 rpm at room temperature for 10 minutes, centrifuged at 15000 g at room temperature for 10 minutes, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction.
[0087] (2) RNA extraction
[0088] (1) Carefully remove the upper layer liquid, add isopropanol drop by drop, shake slowly at room temperature 200rpm for 15 minutes, centrifuge at room temperature 15000g for 10 minutes, and discard all the liquid.
[0089] (2) Add 1ml of 75% ethanol pre-cooled at 4°C, centrifuge at 15,000 g at room temperature for 10 minutes, blot all the liquid with a vacuum pump, open the centrifuge tube and place it at room temperature for 5 minutes.
[0090] (3) Add 10ul DEPC water to dis...
Embodiment 3
[0098] (1) Processing of tissue samples
[0099] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 3 of Table 1 was added, shaken at 200 rpm for 5 minutes at room temperature, centrifuged at 10,000 g for 20 minutes at room temperature, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction.
[0100] (2) RNA extraction:
[0101] (1) Carefully remove the upper liquid, add isopropanol drop by drop, shake slowly at room temperature at 50 rpm for 20 minutes, centrifuge at 10,000 g for 20 minutes at room temperature, and discard all the liquid.
[0102] (2) Add 1ml of 75% ethanol pre-cooled at 4°C, centrifuge at 10,000°C for 20 minutes at room temperature, blot all the liquid with a vacuum pump, open the centrifuge tube and place it at room temperature for 5 minutes.
[0103] (3) Add 10ul DEPC water to ...
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