User:Meng Xiao He/Notebook/fall08/2008/12/10

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PCR tests

  • use 12-8 protocol on cco12.9B, C: each takes cco5F, cco3R, and just pair of 3 outers, 20ul
  • (update: does not work) use Platinum adaptation of 12-8 protocol: 25ul
Cycling conditions:
  1. 5 min at 94
  2. 30s at 94
  3. 30s @ 61, 57 for 40 cycles
  4. 7min @ 72 (should capture WT cells as well), only 2min for last 12 cycles
  5. 10 min@ 72 to end
  6. hold at 4
61: cbb12.9A, B, C, cco12.9D; 57: cbb12.9D, E, cco12.9A

Plating

on sucrose

  • 1:2 dilution into fresh salt-free-LB, let grow 1hr, then plate 10ul
    • cultures were very thin (may be because had to user rocker- shaker is nonfunctional)
    • cco12.9A grew thick, though

on Gm

  • remaining Gm culture of wash off from filter of Duo