12/7 Platinum PCR results
1KB ladder
- unfortunately, EtBr seems to have run so far up gel that bands < 1kb can't be distinguished
- 1-5 were colonies 1, 3, 8, 9, and 10 from the 11-30 "new cco" KO
- then ladder
- next 3: cbb from brown cultures
- next 4: cco from brown cultures
- colonies 1, 2, and possibly 4 have at least taken up the KO plasmid, even if it hasn't been removed from the genome, since the cco flank is itself 1.5 kb
- these conditions work for platinum colony PCR for cco, at least
12/8 Phusion PCR results
Within each block (end with ladder): cco 3R > cco 5F > Duo 3R > Duo 5F > cbb 3R > cbb 5F
Blocks go: (new filter colony 1 > 2 > 3 > 4 > 5 > 6) > old colony > wt control
- for Duo, old and new colony 6 flipped
- for cco, new 5, 6 are actually 1, 2 from 11-30 Gm sensitive, sucrose R colonies ("new cco" KO)
Samples with bands rerun at higher res
- Duo colonies (3R, then 5F): new 1, new 4, old 19, new 6, ctrl
- cbb colonies (3R, then 5F): new 2, new 3, new 4, new 5, new 6, ctrl
- cco colonies (3R, then 5F): new 1, new 2, [... (others had no bands)]
Conclusions
- program used (12/8 on big room machine) seems to work (at least some of the time) with the outer primer sets (in this case, all 3 were added)
- cbb colonies of interest: new 2, new 3, new4, new 5, new 6
- cco colonies of interest: new 1, new 2
Insertional colony processing
- restreaked on LB Gm plates
LB minus salt:
- Duo new # 1 (rename to Duo12.9A)
- cco new # 3 (rename to cco12.9A)
supplemented with 20ul ferric citrate
- Duo brown # 1 (rename to Duo12.9B)
- cco new # 5 (rename to cco12.9B)
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