Dunn School: Notebook 16, Feb 2000-Nov 2000
Influenza related experiments:
- A NP DI (about 420 nt) giving an abnormally truncated NP of 52 amino acids (experiment 616).
- Successful sequencing of eleven independent clones derived from full length band 8 cloning, ranging from 9-17 long mRNA snatched sequences (experiment 620). Not published.
- Unsuccessful attempts to detect "hybrid transcripts" with a ppp 5' end and poly (A) 3' end.
- Unsuccessful experiments using pppA (32P labelled in γ position) to study cDNA transcripts of 49 mer RNA.
- Start of transient transfection (lipofectamine) of 293T cells with 4 cDNA plasmids (for PB1, PB2, PA & NP) to obtain crude recombinant flu polymerase expressed in 293T cells (experiment 633) (experiment 649, very encouraging) (also experiment 641, using GTP label) copying of vRNA 49-mer.
- Transient transfection of 293T cells with mutant PA (post doctoral researchers Ervin Fodor and Mandy Crow) (experiment 650).
- First experiments (looked hopeful) with short synthetic primers of 5' (15 mer) and 3' end (14 mer) of vRNA promoter and transfected nuclear extracts with GTP labelled transcript on 18% gel (experiment 653).
- Heat inactivation studies of PA mutants (from Ervin Fodor and Mandy Crow) of reconstituted recombinant polymerase (experiment 656, 660, 661, 667).
- Start of initially unsuccessful experiments to test for endonuclease activity of recombinant polymerase (e.g. experiment 668 with Mike Leahy, post-doctoral researcher).
- Feb 2000-Nov 2000