Dunn School: Notebook 12, Aug 1994-Oct 1994
All experiments are influenza related. George Brownlee states: "On the whole a very successful set of experiments using in-house synthesized RNA (by Keith Gould)":
- Continuation of notebook 11 (experiment 378) characterization of 5' phosphorylated oligos, particularly presumed (pppoligo 202) although this turned out to be only partially phosphorylated to a mixture of mono- and di- phosphorylated oligos (see below).
- Capping (e.g. experiments 389, 399, etc.) to radiolabel the phosphorylated oligos worked well. These were then used in transcription reactions with micrococcal nuclease-treated influenza cores (a source of flu RNA polymerase).
- Characterization of phosphorylated oligos (P1 nuclease and T2 nuclease digestion, e.g. experiments 402, 405, and by sending samples (desalted and eluted from gels) to McCloskey, Salt Lake City on Robin Aplin's (Oxford, Chemistry) advice. In both cases it was concluded that a mixture of mono and di-phosphoylated oligos were present. No triphosphoryated oligo detected (experiment 411).
- Good transcription with labelled capped and MN cores in vitro (see insert sheet 19 (experiment 431) at end of notebook).
- Experiments with a tied panhandle 50-mer (experiment 428, not phosphorylated) showed incomplete transcription (no full-length transcript) suggesting a block to transcription. No evidence of poly (A) addition. But primer-independent transcripts observed. (see later evidence in Notebook 13).
- Aug 1994-Oct 1994