User:Meng Xiao He/Notebook/fall08/2008/12/09: Difference between revisions
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== | ==12/7 Platinum PCR results== | ||
1KB ladder | |||
[[Image:12-5_pcr_strips.jpg]] | |||
* 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) | |||
[[Image:12-5_pcr.png]] | |||
===Remainder of samples with bands rerun at higher res=== | |||
[[Image:Hires.png]] | |||
*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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Revision as of 10:16, 12 December 2008
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12/7 Platinum PCR results1KB ladder
12/8 Phusion PCR resultsWithin 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
Remainder of samples with bands rerun at higher res
Conclusions
Insertional colony processing
LB minus salt:
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