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Notebook of Joyce Huddleston, Jan 1981-Jun 1981

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MS. 12364/27

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Huddleston's other 4[?] notebooks were (as Brownlee states) unfortunately discarded when she retired in 2008.

Huddleston was Brownlee's research assistant from early 1981, having previously been assistant to Sir Edward Abraham. Brownlee states: "I had to teach her molecular biology. She taught me to keep a better lab notebook."
  1. Brownlee started Huddleston on a new project of preparing full length cDNA clones, in E. coli, of (initially) bands 1-3 of the influenza virus A/NT60/68. He thought they would make an interesting sequence comparison with the progress already being made by Greg Winter and Stan Fields, who continued working on the A/PR/8/34 virus that Brownlee had started in Cambridge.
  2. Over the six months of the project the team tried many steps of optimization. The main strategy was to isolate the full length cDNA of the various A/NT60/68 bands by preparative polyacrylamide gel electrophoresis using a "universal primer" for cDNA synthesis (12-mer). After gel elution they were then back-copied with another universal primer (13-mer) to give dsDNA. cDNA synthesized bands were detected by autoradiography for incorporated 32P (e.g. experiment 19) (bands 1-3 were taken together as they did not separate well) and subsequently back-copied to give dsDNA.
  3. dsDNA was cloned into blunt-ended PvuII-cut pBr322, transformed into competent E. coli, and positive clones selected by Amp resistance.
  4. The first successful cloning may be (GGB) Notebook 1 (experiment 25) in which bands 1-3 clones were obtained, although they appear to have originated from a Joyce Huddleston (JAH) experiment (experiment 19?).
  5. Other bands were then cloned including bands 4, 5 and 6 (e.g. experiments 64, 83, 87, 88) (growth and yield of plasmids in HB101 was better than in X1774). Band 6 clones given to David Bentley (DRB) (page 266) to sequence.
  6. Summary: A very significant six months generating full-length clones from A/NT60/68 virus.

Dates

  • Jan 1981-Jun 1981

Extent

1 volume

Shelfmark

MS. 12364/27

Repository Details

Part of the Bodleian Libraries Repository

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Weston Library
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Oxford OX1 3BG United Kingdom