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●●●●●　RNA polymerase assay　●●●●●



This is a generalized version of the RNA poplymerase assay described for methanogens. The concentration of KCl and the incubation temperature may need adjusting, depending on the characteristics of the RNAP to be detected.

  1. Materials

    1. 0.2M Tris, pH8

    2. 0.1M MgCl2

    3. 1M KCl

    4. 1mg/ml poly d(A:T) - Boeringer

    5. NTP mix
      500ul 10mM UTP 20mM
      50ul 20mM ATP 1mM
      20ul 3H-ATP
      430ul DEPC-treated ddH2O

      [2,8-3H]ATP - ICN #24009 32Ci/mmole in 50% EtOH, 1mCi/ml
      Final ATP sp. activity = 20mCi/mmole

    6. 5% TCA
      1/20 from a 100% stock sol'n (500g + 227ml ddH2O)

  2. Methods
    1. Make up enough of the following solution for n+1 reactions, where n is the number of fractions to be assayed:
      45ul ddH2O
      10ul 0.2M Tris, pH8
      10ul NTP mix
      10ul 0.1M MgCl2
      5ul 1M KCl
      10ul 1mg/ml poly d(A:T)

    2. Mix & divide into 90ul aliquots into 1.5ml eppendorf tubes (95ul aliquots for sucrose gradient fractions).
    3. Add 10ul pol fraction to each tube and incubate at 37C for 20 minutes.
    4. Add 900ul 5% TCA (ice cold) & put on ice for at least 15 minutes.
    5. Filter through glass filters (presoaked in 5% TCA), wash with 2 X 10ml ice-cold 5% TCA, then 2 X 95% ice-cold EtOH.
    6. Dry the filters under a heat-lamp, & count in 5 - 10ml scintillation fluid.

M. Thomm, personal communication
Brown NAR 16:135

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