Notebook:Tk/2008/04/20

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Contents

Transformation results

  • 73 transformants from last week's transformations
  • Most from transformations with low cell density
  • Most from transformations with 10 ul 1M sorbitol added prior to electroporation
  • nothing shows at 14 hours or 24 hours
  • Some colonies visible at 38 hours, more at 48, final at 60 or so

New standard transformation protocol

  • culture cells in 45 ml 1161 medium to light yellow/slightly turbid
  • Spin down and resuspend 3x with 45 ml EPB (no glycerol) on ice in chilled centrifuge
  • Resuspend in 5 ml EPB on ice
  • Aliquot 50 ul cells + 1 ul transposon DNA on ice
  • Add 10 ul 1 M sorbitol
  • Electroporate at 1.5 KV in 1 mm cuvette
  • Immediately add 850 ul 1161 medium, place on ice
  • Incubate 1 hour at 30C in the cuvette
  • Plate 300 ul on 15 ug/ml TET plates
  • Hold remainder in the cuvette for several days at 4C

Newly diluted transposase

  • Dilute 50 ul transposase (8 ug/ul) into 800 ul transposase storage buffer
  • Store at -20, label 500 ng/ul transposase

New transposomes

  • 20 ul transposon DNA (83 ng/ul)
  • 20 ul glycerol (use cut off tip for pipetting)
  • 40 ul diluted transposase
  • incubate 1 hour at 37C
  • hold at -20C

New transformations

  • standard protocol
    • use 10 ul water, 5 ul water, no addition, 5 ul, 10 ul, and 15 ul sorbitol (1 M) additions
  • Test for both positive and negative osmotic pressure changes, and narrower range than last time
  • Plate out all of sorbitol added versions (3 plates)
  • Plate out only 1 plate from water and no addition plates, save remainder at 4C
  • Plates labeled with -10, -5, 0, +5, +10, +15 for water and sorbitol additions (ul added)

New plates and culture medium, standard protocol

Plans for genetics

  • Promoters
    • constitutive
    • heat shock
  • Reporters
    • YFP, mCherry
  • antibiotic
    • cat gene
  • recT recombination gene
  • Test constitutive and heat shock promoters with reporter
  • Test cat gene with constitutive promoter
  • Combine heat shock promoter with recT gene
  • Test recombination efficiency
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