- Gradient cycling temperatures (left to right): 55.4, 56.8, 58.0, 58.9
- 2:15 extensions
- min at 94 for initial denaturation
cbb gradient, cco
- 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
- 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?
- 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
- Does the sucrose selection have to be actively growing cells?
- Do control colony PCRs on any potential "KO" cells
- Sequence the wm plasmid minipreps