User:Meng Xiao He/Notebook/fall08/2008/11/25

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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?)

Image:11.25.pcr.mx.jpg

  • 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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