User:Meng Xiao He/Notebook/fall08/2008/12/07

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Platinum PCRs

Products will be sequenced if any

  • redo of ones that gave 500/1kb bands from 11/30 cco KO's: colonies 1, 3, 8, 9, 10
  • brown cco: 3 from supernatant and 1 from pellet
  • brown cbb: 2 from supernatant and 1 from pellet

25ul reactions, 40 cycles:

  • 7 min initial 94C denaturation
  • 30 s denature
  • 30 s annealing 58→56.5 in first 15 cycles by dropping 0.1 decrease/cycle, 59→56.5 in last 25 cycles
  • 2 min elongation
  • 10 min final extension

conjugation, round 2

  • no MR-1 colonies yet on plates off filter

KO thoughts

  • maybe should supplement with ferric citrate?
    • new LB/no salt cultures for new cco, cbb#3, and Duo#18, supplemented with 500ul of filtered ferric citrate solution (in 8mL salt free LB, concentration unknown...)
  • can it/ need it be done under anaerobic conditions?