User:Meng Xiao He/Notebook/fall08/2008/12/08

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conjugation filter colonies

 * many on cbb and Duo, 4 on cco
 * obvious E. coli growth as well

PCRs to determine proper insertion of suicide vector
Phusion used
 * Colonies:
 * WT, 1 of the old ones ("new cco", cbb3 #3, Duo #19), and 6 new ones
 * cco doesn't have 6 new ones, so used 1 and 2 from 11/30 sucrose selection
 * For each colony: 2 primer sets [flank outer+ 3R] and [flank outer+5F] (note all 3 flank outer sets were included- should be OK? sizes are 4.5, 5.5, 6.5 kb)
 * Cycling conditions (program is 12-8):
 * 1) 2min at 98
 * 2) 7s at 98
 * 3) 30s @ 63->57 in 15 cycles (drop 0.4 degrees per cycle)
 * 4) 30s @ 59->56 in 15 cycles (drop 0.2 degrees per cycle)
 * 5) 30s @ 57 for 10 cycles
 * 3:30 @ 72 (should capture WT cells as well). 45s for last 10
 * 1) 5 min@ 72 to end
 * 2) hold at 4


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