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        Library cDNA Synthesis

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        1064

         

        Library cDNA Synthesis

        1° cDNA Synthesis

        N.B: During 1° cDNA synthesis, all steps should be carried while wearing gloves and all solutions should either have been DEPC-treated or purchased specifically for use with RNA.

        You will need for 1° cDNA synthesis:

        5 x SuperScript RTase buffer (Gibco-BRL)
        5mM methyl dNTPs (Pharmacia, substitute 5m dCTP for dCTP)
        100mM DTT (Gibco-BRL)
        oligo dT primer/adaptor
        [a-32P] dATP (~400Ci/mmol, Amersham or DuPont)
        Ribonuclease inhibitor (RNasin, Pharmacia)
        Sterile, autoclaved, DEPC-treated water
        SuperScript RTase (or SuperScript II, Gibco-BRL)

        You will need for 2° cDNA synthesis:

        10 x DNA Polymerase Buffer (1M HEPES, pH 7.6, 40mM MgCl2 , 2.5mM DTT, 675mM KCl)
        100mM DTT (Gibco-BRL)
        5mM dNTPs (Pharmacia)
        Sterile, nano-pure H2 O
        [a-32P] dATP (~400Ci/mmol, Amersham or DuPont)
        RNase H (Gibco-BRL)
        DNA Polymerase I (Pharmacia)
        3M sodium acetate, 100mM magnesium acetate, pH5.2
        Absolute ethanol
        80% ethanol

        After isolation of mRNA, 1° cDNA synthesis is performed in the following way :-

        1) Heat mRNA to 70°C for 10 minutes and snap chill on ice.

        2) Add, in the following order, to a sterile, RNase-free, eppendorf tube:-

        8ul 5 x SuperScript RTase buffer
        8ul 5mM methyl dNTPs
        4ul 100mM DTT
        2ul oligo dT primer/adaptor (4ug)
        1ul [a-32P] dATP (~400Ci/mmol)
        1ul RNasin
        5.5ul H2 O
        10ul mRNA
        0.5ul SuperScript Reverse Transcriptase (~100 units).

        Mix, incubate at 37°C for 1 hour and stop reaction by chilling on ice. Remove 4ul for analysis of the quantity and quality of the primary cDNA reaction products.

        2° cDNA Synthesis

        To the remaining 36ul of primary cDNA reaction products, ON ICE, add the following in the stated order:-

        40ul 10 x DNA Polymerase Buffer
        15ul 100mM DTT
        12ul 5mM dNTPs
        293ul sterile H2 O
        1ul [a-32P] dATP (~400Ci/mmol)
        1ul RNase H (0.9 units)
        2ul DNA Polymerase I (20 units)

        Mix thoroughly and incubate for 1 hour at 14°C followed by 1 hour at room temperature (22°C). Place reaction on ice.

        Extract once each with an equal volume of phenol/chloroform and chloroform. Precipitate cDNA products by the addition of 40ul 3M sodium acetate, 100mM magnesium acetate, pH 5.2 and 1ml absolute ethanol followed by incubation at -20°C overnight.

        Pellet cDNA by centrifugation at 13,000 rpm at room temperature for 30 minutes. Wash pellet, VERY GENTLY, with 80% ethanol and vacuum dry. Re-dissolve pellet in 44.5ul sterile H2 O and remove 4ul for analysis of cDNA quantity and quality.

        The remaining 40.5ul is ready for blunt-ending.

         

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