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    Home > Biochemistry News > Biotechnology News > TRIzol RNA extraction method.

    TRIzol RNA extraction method.

    • Last Update: 2020-10-17
    • Source: Internet
    • Author: User
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    (i)
    Reagents
    1.
    1. TRIzol reagents.
    2. Chloroform
    3. Isopropyl alcohol
    4. 75% ethanol (DEPC H2O preparation)
    5. DEPC H2O
    (II) Operation step
    1. Sample treatment:
    (1)
    culture
    cells: harvest cells 1-5×107, move into 1.5 ml
    centrifuge tube
    , add 1 ml Trizol, mix well, set at room temperature 5min.
    (2)
    tissue
    : take 50-100 mg of tissue (fresh or -70 degrees C and liquid nitrogen preserved tissue can be) placed in 1.5 ml centrifugal tube, add 1 ml Trizol full homogeneity, room temperature sit 5min.
    2. Add 0.2 ml chloroform, oscillate 15s and set aside 2min.
    3. Centrifuges at 4 degrees C, 12000g× 15min, cleared.
    4. Add 0.5 ml of isopropyl alcohol, gently mix the liquid in the tube and set aside at room temperature for 10min.
    5. Centrifuges at 4 degrees C, 12000g× 10min, discarded.
    6. Add 1 ml of 75% ethanol and gently wash the precipitation. 4 degrees C, 7500g×5min, discarded.
    7. Dry and add the appropriate amount of DECC H2O dissolved (65 degrees C solubility 10-15min).
    (iii) Precautions
    1. Sample volume and Trizol addition must be in accordance with step (1) ratio, can not arbitrarily increase the sample size or reduce trizol volume, otherwise the endo endo-rooted RNase inhibition is incomplete, resulting in RNA degradation.
    2. The experimental process must strictly prevent contamination of RNsae.
    total RNA quantitative
    . The RNA quantification method is similar
    the
    of dna. RNA has the largest absorption peak at a wavelength of 260nm. As a result, RNA concentrations can be measured with 260nm wavelength dlight, with an OD value of 1 equivalent to about 40 μg/ml of single-stranded RNA. If you use a 1cm optical diameter, dilute the DNA sample with ddH2O n times and ddH2O as a blank control, according to the value of OD260 read at this time can calculate the concentration before the sample dilution:
    RNA (mg/ml) - 40×OD26 The ratio of OD260/OD280 for 0 readings × dilution multiply (n)/1000
    RNA pures is 2.0, so the purity of RNA can be estimated based on the ratio of OD260/OD280. A lower ratio indicates the presence
    residual
    protein, and a high ratio indicates degradation of RNA.
    .
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