Superose 6 10 300

The SNR20 91W promoter and its variant SNR20 31D promoter that lacks the region from site −68 to site −7 were previously described (Murakami et al., 2015a (link)). Both promoter fragments (−122/+97) were amplified by PCR and purified using Superose 6 10/300 (Cytiva) in buffer 300. To assemble PIC on templates, the following were mixed in 5 μL of buffer 300: 0.26 μM DNA template, 0.4 μM TFIIA, 1.2 μM TFIIB, 2.4 μM TBP, 0.6 μM TFIIE, 1 μM TFIIF, 0.4 μM coreTFIIH, 1.04 μM pol II, 0.4 μM Sub1 and TFIIK (or Tfb3ΔC mutants) with given amount described above. The mixture was then diluted by adding an equal volume of buffer 10 (20 mM Hepes (pH 7.6), 10 mM potassium acetate, 5 mM magnesium sulfate, 5 mM DTT) and incubated on ice for 24 hours. Run-off transcription reaction was initiated by adding 10 ul of 2x NTP solution consisting of 1.6 mM ATP, 1.6 mM CTP, 1 mM UTP, 1.6 mM GTP, 44 nM [α-³²P] UTP (33 μCi), 10 mM magnesium acetate, and 0.5 U/μL RNaseOUT in buffer 10. Transcription was carried out for 30 min at 30°C then stopped by adding 190 ul of stop buffer containing 300 mM sodium acetate (pH 5.5), 5 mM EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml of proteinase K (Sigma). Transcripts were precipitated by adding 700 ml of ethanol, dried, and analyzed by a denaturing 6% acrylamide gel.