User:Meng Xiao He/Notebook/fall08/2008/11/22

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PCR of suicide vectors

  • ssTOPO A
  • sscco
  • sscbb
  • ssDuo
  • 100 bp ladder
  • PCR with KOentire primer pair:
    • Duo
    • cbb3
    • cco

MR-1 KO electroporation plates

  • No growth (DNA used was ssPCR + template (fair bit)):
    • original transformations
    • ones plated after (night of selection free growth → 4hrs of selection w/ Gm)

WM3064 transformants of KO vectors

  • All are indeed DAP-
  • Need to sequence to ensure whole plasmids transformed
  • Glycerol stocks made

Conjugation attempt

  • Approach 1
  1. Pass 4mL 10mM MgS04 with small volume of MR-1 and WM3064 strain thru. syringe filter
  2. Attach to new syringe w/ 1-2 mL LB DAP
  3. Drip some LB out, so flows thru. entire filter
  4. Cap with either Luer-cap from midi kit or syringe cap
  • Approach 2
  1. Mix o/n cultures of MR-1 and WM3064 strain
  2. Pellet cells
  3. Remove LB and add 4mL 10mM MgSO4 (tried: complete resuspension with Duo, and partial resuspension with cco and cbb)
  4. Pellet cells
  5. Replace MgSO4 w/ LB DAP

Both left in beaker in 30C incubator

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