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文献和实验Polymerase III in vitro Transcription
: 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
PolymeraseIII invitro Transcription
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 H2 O to a final volume of 16 microliters less the maximum
Sucrose Density Gradient Fractionation of Yeast Membranes
to eppendorf tube, centrifuge twice (spin once then re-spin sup) at 500 X g. for 10 min., 4oC Remove 100 ul of sup as "input." add 100 ul 2X SDS-PAGE sample buffer. Put in 100oC heat block for 10 min., then transfer to -20oC. Add 650 ul
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