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?


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