Conjugation results
Filter
- cbb3, plate: >100 colonies, although sizes vary greatly, with many large
- cbb3, liquid: MR-1 growth (from pellet color)
- cco, liquid: looks like E. coli is growing? Pellet is white
- Duo, plate: 2 colonies, medium sized
- cbb3 and Duo, filter into liquid: both cultures thick, streaked for isolation on LB Gm plates
- cco: no growth in filter+liquid yet, plated cultures had many (~50) white, colonies, that look like E. coli
Spin down
- cbb3 streaked: scattered colonies at beginning of streak
- cbb3, liquid: MR-1 growth (from pellet color)
- cco, liquid: white pellet > E. coli?
- Duo streaked: 2 colonies
- plating cell mix: this seems like a back idea, as significant amounts of E. coli growth occurs in patches (not isolated colonies)
Colony PCR
Used Platinum Taq mix, 10min 94C initially cell boiling (maybe too much?)
- No colony from conjugation gave bands, just smears as in first few lanes.
- last 4 lanes: cbb3 WM plasmid miniprep PCR, Duo WM plasmid miniprep PCR, cco WM plasmid miniprep PCR
- Duo doesn't seem to work at this annealing temp (56C)
- colony PCR needs protocol change
Next
- cco and new MR-1 grown up for new try w/ filter system
- 2 colonies of Duo and cbb each grown up in LB, LB Gm, and on sucrose plate
Transformation update
- Old transformations (last set that had no intervening nonselective growth period) developped yellow-orange contaminant colonies. Clearly not E. coli/MR-1 b/c growth was minimal overnight. Plates discarded.
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