User:Meng Xiao He/Notebook/fall08/2008/12/09

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12/7 Platinum PCR results
1KB ladder


 * unfortunately, EtBr seems to have run so far up gel that bands < 1kb can't be distinguished
 * flecks are from bubbles
 * some of the first few samples didn't seem to have left well?
 * 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)



Remainder of 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)→ambiguous b/c cannot see if the WT 5.4kb cbb outer fragment is indeed missing]
 * new4, new 5 don't look like they incorporated the vector, since the WT 5.4kb cbb outer fragment is still there- what are those bands btw 1.6kb and 2kb?
 * cco colonies of interest: new 1, new 2 (a little iffy> is there still the 6.5 kb WT cco outer fragment?)

Insertional colony processing

 * restreaked on LB Gm plates

LB minus salt:

 * cco new # 1 (rename to cco12.9A)
 * cbb new # 3 (rename to cbb12.9A)


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