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        mRNA Purification

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        1310

        mRNA Purification

         


          I. Prepare oligo-dT cellulose

        • use 40 mg oligo-dT cellulose / 1 mg total RNA
        • swell oligo dT-cellulose in elution buffer
        • wash oligo dT-cellulose 4 x with elution buffer (30 sec. full speed spin in between)
        • equilibrate oligo-dT cellulose with 2 to 3 washing steps using 1x binding buffer

         II. mRNA purification

        • bring 1 mg total RNA with H 2 O to 600 µl
        • incubate 4 min at 65 °C
        • add 600 µl 2x binding buffer
        • add to 40 mg 1x binding buffer equilibrated oligo-dT cellulose
        • incubate 15 min at RT on a rolling incubator or vortex several times in between
        • spin oligo-dT cellulose down, discard supernatant
        • wash 2 x with 1x binding buffer
        • wash 2 x with wash buffer
        • elute with 250 µl elution buffer at 37 °C for 5 min
        • spin and keep supernatant (for second round of purification or precipitation)
        • elute oligo-dT cellulose again with 250 µl elution buffer
        • spin and keep supernatant (for second round of purification or precipitation)
        • combine eluate and add H 2 O to 600 µl
        • repeat mRNA purification by starting again with 4 min incubation at 65 °C

         III. Recover mRNA

        • add 50 µl 4 M NaCl and precipitate with 2 vol. cold EtOH
        • incubate 1 h at -20 °C
        • spin 10 min full speed, wash with 70 % EtOH, air dry and dissolve in 20 µl H 2 O
        • read A260 of 1 µl (100 to 250 fold diluted)
        • run 1 µg ( and 10 µg total RNA as comparison) on a 1.2 % agarose Formaldehyde gel

          recovery = 1-2 % of the total RNA (10-20 µg mRNA / 2 mg total RNA)

         


        Buffers:
        [all buffers should be made with DEPC treated/autoclaved components!]

         oligo dT cellulose (type7, Pharmacia)

         2 x binding buffer:
        1 M NaCl; 20 mM Tris pH 7.5; 2 mM EDTA; 0.1 % SDS
        [50 ml : 12.5 ml 4 M NaCl; 1 ml 1 M Tris; 200 µl 0.5 M EDTA; 500 µl 10 % SDS]

         1 x binding buffer:
        0.5 M NaCl; 10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS
        [50 ml: 6.25 ml 4 M NaCl; 500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS]

         wash buffer:
        0.2 M NaCl; 10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS
        [50 ml: 2.5 ml 4 M NaCl; 500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS]

         elution buffer:
        10 mM Tris pH 7.5; 1 mM EDTA; 0.05 % SDS
        [50 ml: 500 µl 1 M Tris; 100 µl 0.5 M EDTA; 250 µl 10 % SDS]

         

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