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        RT-PCR Analysis--详细的RT-PCR方法

        互联网

        1233

        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 72m l b ME

        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 b ME 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.

         

        • Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.

         

        • Resuspend the pellet in 300 m l Lysis Solution and add 300 m l isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.

         

        • Resuspend the pellet in 10 m l DEPC treated Q and store at -80°C.

         

        reverse transcription reaction

        • Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml).

        • Heat to 65° for four minutes and immediately transfer to ice.

        • Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:

        2 microliters 10X RT buffer

        1 microliter 1 mg/ml BSA

        1 microliter 20 mM DTT

        0.5 microliters RNaseIN

        0.4 microliters 25 mM dNTPs

        0.2 microliters AMV RT

        9 microliters Q

        • Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°.

        pcr reaction

        • Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction.

        • Add the following PCR cocktail per tube:

        2.5 microliters 10X PCR buffer

        1.5 microliters 25 mM MgCl2

        1 microliter each primer (10pmol/microliter; 55° Tm)

        0.2 microliters 25 mM dNTPs

        0.025 microliters a -32 P dATP

        0.02 microliters Taq polymerase (protocol T.2)

        14 microliters Q

        • Perform PCR using the following cycle parameters:

        94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes.

        • The number of cycle should be determined empirically to find the linear range of amplification.

        <center> <p>  </p> </center>
        上一篇:Long PCR Reagents and Guidelines   下一篇:反转录生产cDNA链以及PCR扩增
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