Notebook of Joyce Huddleston, Jan 1981-Jun 1981
File
MS. 12364/27
Held in our offsite storage facility
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."
- 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.
- 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.
- dsDNA was cloned into blunt-ended PvuII-cut pBr322, transformed into competent E. coli, and positive clones selected by Amp resistance.
- 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?).
- 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.
- Summary: A very significant six months generating full-length clones from A/NT60/68 virus.
Dates
- Creation: Jan 1981-Jun 1981
Extent
1 volume
Language of Materials
- English
Shelfmark
MS. 12364/27
Repository Details
Part of the Bodleian Libraries Repository
Contact:
Weston Library
Broad Street
Oxford OX1 3BG United Kingdom
specialcollections.enquiries@bodleian.ox.ac.uk
Weston Library
Broad Street
Oxford OX1 3BG United Kingdom
specialcollections.enquiries@bodleian.ox.ac.uk