User:Meng Xiao He/Notebook/fall08/2008/11/27

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PCR results

  • Gradient cycling temperatures (left to right): 55.4, 56.8, 58.0, 58.9
  • 2:15 extensions
  • min at 94 for initial denaturation
  • Platinum

cbb gradient, cco

11-27 gel1.mx.jpg

  • 1-4: cbb3 plasmid (wm)
  • 5-8: cbb3 MR-1 conjugant colonies 1-4
  • 1kb ladder
  • cco MR-1 conjugant colonies 1, 2
  • ignore last, wrong setup

Duo gradient, WM miniprep Sma digests

11-27 gel2.mx.jpg

  • 1-8: plasmid and then colony PCR of Duo
  • 1kb ladder
  • SmaI digests: Duo, cbb, cco

A little confusing

  • SmaI digests seem to have failed, as previous PCR (11/25) clearly shows that cbb and cco plasmids are correct size and pure
    • It is the Duo plasmid has completely failed, as it has not PCRed successfully. This would suggest that MR-1 can spontaneously develop Gm resistance? Yet did not occur with the cco conjugation. This also conflicts with the apparent sensitivity of the Duo transconjugants to sucrose.
  • cbb PCR is very messy. At 56C (see 11/25) it was pretty clean though, if a bit faint. Also, this suggests that the conjugation did work, but the colonies were not sucrose sensitive?

Next steps

  • New cco conjugation attempts were plated on LB Gm plates
  • Duo 17 and 18 grown o/n on LB then o/n on LB minus salt were plated on LB sucrose minus salt
  • cco MR-1 colony 1 looks like it has a faint band at the 2kb mark, so grown up (for o/n) in LB-salt
  • cbb colony 3 selected for o/n culture in LB-salt

Figure out

  • Does the sucrose selection have to be actively growing cells?
  • Do control colony PCRs on any potential "KO" cells
  • Sequence the wm plasmid minipreps