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
  6. 3:30 @ 72 (should capture WT cells as well). 45s for last 10
  7. 5 min@ 72 to end
  8. hold at 4
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