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        Nuclear enrichment

        互联网

        1208

        实验试剂

         

        1. Buffer A

            10mM HEPES

            1.5mM MgCl2

            10mM KCl

            0.5mM DTT

            0.05% NP40 (or 0.05% Igepal or Tergitol)

            pH 7.9

        2. To prepare 250ml stock of Buffer A:

            HEPES: 1M = 238.3g/litre, therefore 10mM = 0.59g/250ml

            MgCl2 : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

            KCl: 1M = 74.5g/litre, therefore 10mM = 0.187g/250ml

            DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

            NP40 = 0.05% (v/v)

        3. Buffer C

            5mM HEPES

            1.5mM MgCl2

            0.2mM EDTA

            0.5mM DTT

            26% glycerol (v/v)

            pH 7.9

        4. To prepare 250ml stock of Buffer C:

            HEPES: 1M = 238.3g/litre, therefore 5mM = 0.295g/250ml

            MgCl2 : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

            EDTA: 1M = 372.2g/litre, therefore 0.2mM = 0.0186g/250ml

            DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

            26% Glycerol (v/v) = 65ml

            4.6M NaCl

            87.66g/326ml H2 0

        实验步骤

         

        1. Requires large 12ml petri dishes. Prepare 1ml of Buffer A with added cocktail of protease inhibitors from a frozen stock and store on ice.

        2. Add 500µl of Buffer A per large petri dish on ice and scrape thoroughly, leave on ice for 10 min (NP40 primarily lyses plasma membrane and leaves other membranes more intact than Triton X-100 does).

        3. Centrifuge at 4o C at 3000 rpm for 10 min.

        4. Remove supernatant and keep (this will contain everything except large plasma membrane pieces, DNA, nucleoli), extract out 10µl for Bradford assay.

        5. On ice, resuspend pellet in 374µl of Buffer C and add 26µl of 4.6M NaCl to give 300mM NaCl (high salt helps lyse membranes and forces DNA into solution).

        6. Homogenise with 20 full strokes in Dounce or glass homogeniser on ice.

        7. Leave on ice for 30 min

        8. Centrifuge at 24,000g for 20 min at 4o C

        9. Aliquot supernatant, remove 10µl for Bradford assay and store at –70o C.

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