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        半定量RT

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        1885
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        RT-PCR Analysis

        Solutions

        10X RT Buffer

        10X PCR Buffer

        100 mM Tris pH 9.0

        500 mM KCl

        1% Triton X-100

        25 mM MgCl2

        use at a concentration of 1.5 mM

        Lysis Solution

        4M GuSCN 250 g guanidine thiocyanate

        25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0

        0.5% Sarkosyl 26.4 ml 10% Sarkosyl

        add 293 ml Q

        before use, add 72ml bME

        Water Saturate Phenol

        thaw 500 ml phenol

        add 0.5 g hydroxyquinolin

        add 500 ml Q

        mix and allow phases to separate at room temperature

        repeat 2 times and store at 4°C

        3M NaOAc 5.2

        24.6 g NaOAc (anhydrous)

        pH to 5.2 with acetic acid

        up to 100 ml Q

        Procedure

        rna isolation

        • Add tissue to 400 microliters lysis solution with bME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80°C.

        • Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform.

         

        • Mix by inversion and hold on ice for 15 minutes.

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