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Dunn School: Notebook 20, Jul 2004-May 2006

MS. 12364/23
Held in our offsite storage facility

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Notebook covers experiments including:

  1. Brownlee continued with large scale (experiment 78 onwards) in vitro primer-independent transcription experiments using cRNP preparations from Tao Deng (DPhil student) and Jane Sharps. Brownlee worked up the reaction before running PAGE gels, and subsequently eluted bands from these gels. Products were further analysed by T1 RNase or pancreatic RNase digestion and 20% gels run, or by various enzymes and analysed by chromatography.
  2. Experiment 79: pppG identified on PEI-cellulose chromatography suggests initiation at position 2 of promoter. Experiment 83 series of pppG and pppA-ended products on T2 RNase and P1 nuclease analysis on PEI chromatography.
  3. Following success with synthesis of primer-independent products with cRNP preps., Brownlee decided to analyse DINUCLEOTIDEs synthesis (with either cRNP or various recombinant polymerase preps, with or without NP expression, with or without added vRNA or cRNA promoter on 25% PAGE urea gels -stimulated by paper from Ishihama’s group. NP did not influence activity (experiment 106).
  4. pppGpC and pppApG identified (experiment 84) and authenticated. pppGU tentatively identified (experiment 108) from cRNA promoter.
  5. Time course suggests synthesis proceeds over a long time (experiment 107).
  6. Synthesis primed by adenosine, AMP, ADP and ATP compared (stimulated by Ishihama's paper) (experiment 85). Synthesis was dependent on both strands of promoter and their base pairing with one another, and integrity of hairpin loop of 5' end of promoter but hairpin in 3' promoter strand was less convincing (e.g. experiments 115, 117, 118).
  7. Native gels (non-SDS, Laemmli gels). SDS treatment tried on Baculovirus expressed PA prepared by Koyu Hara (post-doc). PA appeared to aggregate forming a ladder up to a tetramer (experiment 137). It was mainly 1 band after SDS treatment.


  • Creation: Jul 2004-May 2006


1 volume

Language of Materials

  • English


MS. 12364/23

Repository Details

Part of the Bodleian Libraries Repository

Weston Library
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Oxford OX1 3BG United Kingdom