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    Home > Biochemistry News > Biotechnology News > Separation and identification of nucleic acids in liver tissue.

    Separation and identification of nucleic acids in liver tissue.

    • Last Update: 2020-10-22
    • Source: Internet
    • Author: User
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    related topics 1,
    the separation
    and identification
    of
    DNA
    in liverorganizations1 . Master the principles and methods of DNA separation purification.2 . Be familiar with the
    of desktop centrifuges
    devices..3. Master DNA quantification techniques.
    nucleic acids
    incells exist in the form of nuclear proteins, wherein DNA proteins are mainly present in the nucleation of cells, nuclear proteins are mainly present in cytogenes. The solubility of these two types of nuclear proteins in the 0.14mol / L sodium chloride solution is very different, the ICTs have a high degree of solubility in this solution, but the solubility of the DNA protein is quite low. After the liver has been homogeneed, use 0 . 14mol / L sodium chloride solution is drawn to separate the two nucleoproteins. A small amount of sodium citrate is added to the separation process to inhibit the hydrolyzing of DNA by DNA.
    SDS
    (sodium tyranyl sulfate) enables DNA to dismerge, adding SDS to a solution containing DNA protein, dna is parted from
    protein
    , the protein is precipitated with chloroform, and DNA dissolves in the water phase, and finally uses cold ethanol to dissect DNA and obtain purified DNA. The addition of a small amount of isoquinol to chloroform can reduce the production of the operating process foam and help to split the phase, so that the centrifugal upper water phase, the middle denatured protein, the lower
    align organic
    solvent phase to maintain stability. DNA and RNA have high absorption peaks at wavelengths of 260nm, and proteins have high absorption peaks at 280nm.
    this principle, the absorbence of the purified sample at 260nm and 280nm can be used to calculate the concentration of DNA in the sample and determine its purity. Under standard conditions, the double-stranded DNA concentration in the sample is 50 μg/ml and the single-stranded DNA concentration is 40 μg/ml. The ratio of pure DNA sample A260/A280 is approximately 1.8, and the sample contains proteins or other impurities that reduce the ratio of A260/A280.Equipment and
    Reagents
    ) 1. Equipment Glass homogenizers, centrifuges, test tubes, scale straws, UV-visible phosphorescometers 2. Reagents 1.9% sodium chloride solution 2.10% sodium chloride solution 3. 0.14 mol/L sodium chloride solution (containing 0.01 mol/L sodium citrate): sodium chloride 8.182g and sodium citrate 2H2O 2.941g, dissolved and diluted to 1000m 1 4.95 %Ethanol Solution ( Refrigeration ) 5.5 % Sodium Dixane Sulfonate (SDS) Solution : 25 g SDS Dissolved in 500m 1 45 % Ethanol 6. Chloroform - Isolycol Mixture: Chloroform: Isopropyl Alcohol : 24:1 .7. 0.1 mol/L NaOHSteps:1 . Liver homogenous preparation: fresh pig liver, washed with 0.9 % NaCI blood, removed connective tissue, cut, said to take 4g liver tissue, plus 4 ml 0.14mol / L NaCl solution, abrasive in the homogenizer to make liver slurry.2 . Separation of nuclear protein: liver slurry poured into the test tube, 4000r/min centrifugation 5min, discarded. Precipitation plus 2 m 1 0.14 mol/L NaCl stirred and then ground in a homogenizer, 4000r/min centrifugal 5min, discarded, precipitated repeated, discarded, precipitated as a DNA-protein complex.3 . Add 0.14mol/L NaCl 2.0ml to the precipitation, stir well, drip 5 % SDS 2.0m1, side-by-side stir, 60 degrees C water bath 10 min (non-stop stirring), cold to room temperature, evenly divided into two tubes for Bl, B2. 4 . B1, B2 tube each drop of chloroform - isosterol liquid 4.0m 1 (side-by-side stirring), stirred to the solution color uniform, 3000r/min centrifugation 15min. The solution is divided into three layers, the upper layer is water phase (including DNA), the middle layer is protein precipitation, the lower layer is organic phase, absorbing B1, B2 upper liquid poured into another test tube. 5 . Top liquid plus 95 % cold ethanol 4.0m 1, mixing (inverted mixing method), 3000r/min centrifugation 15min, go on liquid, precipitation for purified DNA. 6 . Dissolve of DNA: 0.1 mol/L NaOH 4.0 ml is added to the precipitation, stirring dissolves, 2000r/min centrifuges 10min, and the upper liquid is DNA hydrolyte. 7 . DNA determination: Dilute the sample to a certain concentration with 0.1mol/L NaOH, zero the sample at 0.1mol/L NaOH, measure the sample's A260 and A280, calculate the DNA content per ng of liver tissue, and determine the purity of purified DNA. Note: 1 . When preparing liver homogenization, try to do so in cold conditions and add 0.14mol/L sodium chloride solution containing 0.0l mol / L sodium citrate as soon as possible to inhibit DNA and prevent DNA decomposition to increase nucleic acid intake. 2 . Each step should strive to be accurate, minimize the loss of mid-course nucleic acid, to ensure the accuracy of quantitative measurement. 3 . After dilution, try to keep samples A260 and A280 in the range of 0.1-0.7. , the separation and identification of RNA in liver tissue "Experimental Purpose" 1 . Master the principle and operation method of RNA separation purification2 . Mastering RNA Quantification Technology Experimental Principles According to the previously described, DNA and RNA are two types of nuclear proteins with different degrees of solubility in 0.14mol / L sodium chloride solution, and after liver homogenization, 0.14mol / L sodium chloride solution is used to separate the two nucleoproteins. In a solution containing 0.14mol / L sodium chloride containing RNA, adjust pH to 4.2 so that it reaches an equal point of electricity, the RNA is precipitated from the solution; the activity of RNase during the separation process to prevent the degradation of RNA. RNase is ubiquitous and high temperature resistant, and RNA extraction conditions are much more stringent than DNA, often using DEPC (carocarbonate) to inhibit RNase activity. The concentration and purity determination of RNA in the sample, the same as before, will not be repeated here, the higher purity of the RNA sample, A260/A280 , 1.8 to 2.0, if A260/A280 < 1.8, indicating that the sample contains protein, etc. , can be removed by phenol- chloroform. Equipment and Reagents1. Equipment Glass homogenizers, centrifuges, UV-visible estrometers, test tubes, scale straws 2. Reagents (1) 0.9 % sodium chloride solution (2) 10 % sodium chloride solution (3) 0.14mol / L sodium chloride solution (containing 0.01 mol / Sodium L citrate): Sodium chloride 8.182g and sodium citrate 2H 2O 2.941g, dissolved and diluted to 1000m 1 (4) 10% acetic acid solution (5) 95 % ethanol solution (refrigeration) (6) 0.1mol/L NaOH (Operation Steps) 1 . Liver homogenous preparation: fresh pig liver, washed with 0.9 % NaCl blood, removed connective tissue, shredded, called 4g liver tissue, plus 4m 1 0.14mol/L NaCl solution, ground in a homogenizer to make liver slurry. 2 . Separation of nuclear protein: liver slurry poured into the test tube, 4000r/min centrifugation 5min, upper clear pour into another test tube for the A tube. Precipitation plus 2m 1 0.14mol / L NaCI stirred and then ground in the homogenizer, 4000r/min centrifugal 5min, upper clear pour into the A tube, precipitation repeat the above operation, upper clear into the A tube, precipitation abandoned. 3 . A tube with 10 % acetic acid to tune pH to 4.2, mix well, set aside 5min, 4000r/min centrifugal 5min, pour on clear, precipitated containing keruclein. 4 . A tube precipitation plus 3m 1 10 % NaCI, stir well, 100 oC water bath 10min (constantly stirring, anti-boiling overflow), cooling, 4000r/min centrifugation 5min, leaving liquid. 5 . A tube on the liquid plus 95 % cold ethanol 5m 1, see lactose sodium RNA precipitation, mixed and placed 10min, 4000r/min centrifugal 15min, precipitation for purified RNA. 6 . Dissolve of RNA: 0.1 mol/L NaOH 4.0ml added to the precipitation, stirring dissolve, 2000r/min centrifugation 10min, and RNA hydrolyte. 7 . RNA assay: Dilute the sample to a certain concentration with 0.1mol/L NaOH, zero the sample at 0.1mol/L NaOH, measure the A260 and A280 of the sample, calculate the RNA content per ng of liver tissue, and determine the purity of the purified RNA. Note: 1 . While preparing liver homogeneity, it is in the process. In the ice bath, all the exercise process attention to inhibit RNase activity. 2 . Each step should strive to be accurate, minimize the loss of mid-course nucleic acid, to ensure the accuracy of quantitative measurement. 3 . After dilution, try to keep samples A260 and A280 in the range of 0.1-0.7. : Why use salt when separating RNA? . 2. How to determine the purity of the dna extracted? . 3. What reagents are usually used to isolate purified DNA
    .
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