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        Pol III whole cell yeast extract (small scale)

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        1126

         

        Pol III whole cell yeast extract (small scale)

        Mike Schultz, Pavel Aprikian, Steve Hahn

        last modified6/28/99

         

        Grow 2 liters yeast in YPD (with 3% glucose) to an A600 of 1.5 to 2.0 (inoculate ~1.5 ml saturated overnight in 1 liter media and grow ~14 hr at 30 degrees). Expect 5-6 g of cells.

         

        Harvest cells and wash cells once in 30 ml cold H2O.

        Wash cells once in 30 ml cold extraction buffer.

        Weigh cell pellet.

        Scrape pellet into a 10 ml syringe and squirt into a 50 ml screwcap tube which is suspended into liquid N2 (the tube can also contain a little liquid N2 if desired).This cell pellet can be frozen at -70 deg. for at least one year with no loss of activity.

        Precool a large (~8 inch diameter) mortar and pestle. Put on dry ice. Add a little liquid N2 to cool.

        Dump in frozen yeast and grind big pieces until everything is uniformly small powder (~ 1min).

        Add a little more liquid N2 being careful to not make powder fly all over.

        Grind again for about 1 min. Repeat liquid N2 addition and 1 min grinding.

        Use a precooled spatula to scrape the powder into a precooled weigh boat. Dump into a 50 ml conical tube. Store tube on dry ice if grinding other yeast cells.

        After all grinding is complete, transfer tube to ice and add 15% the total volume of extraction buffer (i.e 5 g cells + 750 ul buffer) and let sit on ice 20 min. Scrape the yeast sorbet into an ultraclear SW55 tube and spin in ultracentrifuge 100,000 x g for 1 hr at 4 deg. (33,000 rpm in SW55).

        After spinning, ~1/3 volume is cell pellet, ~2/3 volume a clear yellow liquid and a thin white lipid layer on is on top. Remove clear liquid with a syringe being careful to avoid the pellet and lipid layer.

        Dialyze 3 X 1 hr at 4 degrees vs 500 ml dialysis buffer.

        Aliquot and freeze extracts.

        Expect 20-30 mg/ml protein. Typically, use 20 micrograms in a Pol III in vitro transcription reaction.

         


         

        Solutions needed for WCE:

        Extraction Buffer (500 ml)

         

        100 mM HEPES, 7.9 11.9 g HEPES
        245 mM KCl 9.14 g KCl
        5 mM EGTA 10 ml 0.25 M EGTA
        1 mM EDTA 2 ml 0.25 M EDTA
          H20 to 500 ml

         

        Just before use, add protease inhibitors and add DTT to 2 mM.

         

         

         

        Just before use, add protease inhibitors and add DTT to 2 mM.

         

         

        0.1 M PMSF (100x)

        16 mg/ml Ethanol

        Store at -20 degrees

        0.2M DTT

        32 mg/ml H2O

        Store frozen at -20 degrees

        Benzamidine (100X)

        31 mg/ml H2O.

        Store frozen at -20 degrees

        Leupeptin (500X)

        0.15 mg/ml Ethanol.

        Store at -70 degrees for less than 6 months

        Pepstatin (200X)

        0.28 mg/ml methanol

        Store at -20 degrees.

        Chymostatin (2,500X)

        5mg/ml DMSO

        Store frozen at -20 degrees

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