User:Tk/Notebook/MF-xfm/2008/04/11

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Transformations

  • still no transformants
  • E. coli control worked well
    • 1.25 KV control gave 100 transformants (1.75 hr. outgrowth)
    • 1.75 KV control gave 850 transformants (1.75 hr. outgrowth)
  • tried again today with 50 ml culture spun down to 250 ul with 2x wash in EPBG
  • culture was sligntly turbid, yellow
  • Four transformations:
    • plain
    • with 1 ul BSA added (NEB 100x)
    • with 1 ul pUC19 DNA added (1 ug/ul)
    • with both BSA + DNA added
  • electroporation at 1.75 KV, 1 mm gap
  • outgrowth 1.5 hours
  • no sparking
  • cultures turned yellow
  • plated 300 ul
  • also redid E. coli control

Possible problems

  • bad plates -- test growth of older MF resistant strains on the new plates
    • plated earliest cultures A,B,C and wt MF on a tet plate (4/9/08)
  • temperature bad?
    • but normal MF cells grow fine
  • cell concentration still too low
    • obviously much lower than E. coli dense cell growth
  • should also test trypsin treatment