User:Meng Xiao He/Notebook/fall08/2008/12/06: Difference between revisions

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Products will be sequenced if any
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, (11 as + control)
*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 cco: 3 from supernatant and 1 from pellet
*brown cbb: 2 from supernatant and 1 from pellet
*brown cbb: 2 from supernatant and 1 from pellet
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*2 min elongation
*2 min elongation
*10 min final extension
*10 min final extension
==conjugation, continued==
==conjugation, continued==
* cells vortexed off filter and plated on LB Gm
* cells vortexed off filter and plated on LB Gm

Revision as of 14:47, 7 December 2008

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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, continued

  • cells vortexed off filter and plated on LB Gm

brown supernatants

  • cco and cbb KO (in salt-free LB) cultures have turned brown
  • supernatants and pellets cultured
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