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        Polymerase III in vitro Transcription

        互联网

        1609

        Polymerase III in vitro Transcription

        Steve Hahn

        For the following reactions, use appropriate shielding and dispose of radioactive waste properly!

        A 20 microliter transcription reaction contains:

        4.0 μl 5X Pol III transcription buffer

        0.2 μl 0.1 M DTT

        0.2 μl alpha amanatin (1 mg/ml)

        0.2 μl RNAse Inhibitor (Amersham, 20 U)

        2.0 μl NTP stock

        0.5 μl alpha 32P GTP

        140 ng plasmid template (pSK LEU3, pCH6), [use 280 ng 5S pUC template]

        H2O to 20 microliters final volume.

        Transcription Protocol:

        For a Typical transcription reaction, make an NTP mix containing:

        2.0 μl NTP stock

        0.5 μl alpha 32P GTP

        DNA template

        H2O to a final volume of 3 microliters

        Make a second mix containing all other transcription components except protein:

        4.0 μl 5X Pol III transcription buffer

        0.2 μl 0.1 M DTT

        0.2 μl alpha amanatin (1 mg/ml)

        0.2 μl RNAse Inhibitor

        H2O to a final volume of 16 microliters less the maximum volume of protein to be added.

        Aliquot the second mix to all reaction tubes and incubate on ice.

        Add the protein extract (typically 20-40 micrograms of a whole cell extract) and any other proteins to be added. Add extract dialysis buffer (or protein dilution buffer) to bring all reactions to the same volume.

        Add 3 μl of the NTP mix to each reaction, mix by flicking the tube 5-6 times, and immediately incubate at 30 degrees (the reactions will work well between room temp and 32 degrees).

        After 30 min., stop the reaction with 180 microliters stop solution.


        Extract once with phenol/CHCl3 (2/1). ETOH precipitate with 20 μl 3M NaOAc and 600 μl ETOH.

        Freeze 10 min. Spin 10 min. Remove sup. with a drawn out pasteur pipette. Add 200 μl 100% ETOH and spin 3 min. Remove sup. again.

        Dry pellets but do not overdry. Thoroughly resuspend pellets in Formamide dye solution containing 0.1% SDS.

        Heat 1 min. 90 degrees and put on ice. Load to 0.4 mm thick urea acrylamide gel (don’t forget to clean the wells of the gel just before loading). Typically, run 300V, 60 min until the bromphenyl blue is 3/4 of the way to the bottom.

        Dry gel 20-30 min. Typically expose ~3 hr or overnight without a screen.

        Solutions needed for Pol III transcription:

        5X Pol III Buffer (store frozen) (1 ml):

        10% glycerol

        200 ul 50% glycerol

        100 mM HEPES, 7.9

        100 ul 1M HEPES

        400 mM KCl

        400 ul 1M KCl

        25 mM MgCl2

        25 ul 1 M MgCl2

        5 mM EDTA

        20 ul 0.25 M EDTA

         

        255 ul H2O

        NTP Stock (store frozen ―70 deg):

        5 mM each ATP, CTP, UTP

        0.5 mM GTP

        7.3 ml Stop mix (make fresh):

        0.1 M NaOAc

        250 ul 3M NaOAc

        10 mM EDTA

        300 ul 0.25 M EDTA

         

        6.4 ml H2O

        0.5% SDS

        375 ul 10% SDS

        5 ug/ml tRNA

        1.2 ul tRNA (30 ug/ml)

        Extract Dialysis Buffer (store frozen ―70 deg):

        20 mM HEPES, 7.9

        100 mM KCl

        5 mM MgCl2

        1 mM EDTA

        20% glycerol

        2 mM DTT


        Protein dilution buffer (5 ml) store -70 deg:

        20 mM Tris 8.0

        100 ul 1 M Tris 8.0

        1 mM DTT

        50 ul 0.1 M DTT

        10% glycerol

        1 ml 50% glycerol

        150 mM KCl

        0.75 ml 1 M KCl

        1 mM EDTA

        20 ul 0.25 M EDTA

        50 ug/ml BSA

        2.5 ul 100 mg/ml BSA

         

        H2O to 5 ml total volume

        20 ml Acrylamide Urea Gel:

        3 ml 40/2% sequencing acrylamide

        2 ml 10X TBE

        8.5 g Urea

        8 ml H2O

        microwave ~10 sec and stir to dissolve.

        Degas

        Add 175 μl 10% ammonium persulfate

        Remove 1 ml acrylamide and add 1 μl TEMED. Pour plug at bottom of gel. Let polymerize a few minutes.

        To the remaining acrylamide, add 18 microliters TEMED and pour gel. Gel can stored O/N, if kept wet and sealed at room temp.

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