Bacterial Transformation

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Bacterial Transformation
October 3, 2002 Sarah Brockway Microbiology and
Immunology Denison Lab
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Bacterial Transformation

• Introduction
• Aseptic technique
• Reagents
• Cell types
• Preparation of competent cells
• Heat-shock transformation
• Electroporation
• Plating on selective media
• Important tips

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Introduction

• Bacterial transformation is the introduction of
  a plasmid (circular DNA) into bacteria.
• The purpose of bacterial transformation is
  usually to amplify the plasmid DNA.
• Plasmids can also to be introduced into bacteria
  for protein expression purposes.
• Heat-shock transformation and electroporation are
  the two techniques commonly used.

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Aseptic Technique

• Aseptic technique involves avoiding contact of
  the bacterial cultures, sterile medium and
  sterile surfaces of growth vessel with
  contaminating organisms.
• Always wash hands, wear gloves and work on a
  clean surface.
• Flaming the mouth of media bottles, tubes, loops,
  cell spreaders, etc. will avoid contamination.

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Reagents Needed

• Ice, pipettes, sterile tips, tubes
• Water bath (42ºC)and Shaker (37ºC)
• Cuvette and Electroporator
• Competent cells
• Plasmid DNA (ng amounts)
• Nutrient media luria broth (LB) or SOC
• Selective agar media (plates)
• Cell spreader or glass beads

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Cell Types

• E. coli is a gram-negative bacteria used most
  commonly for transformation.
• Lab strains of E. coli are attenuated for
  pathogenicity.
• Common strains include
• -DH5?, BL21(heat-shock)
• -NM522, DH10B (electroporation)
• Cells can be purchased as competent or they can
  be made competent with chemical treatment.

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Prep of Competent Cells

• Chemically competent cells are prepared by
  treatment CaCl2 which renders the cell able to
  uptake DNA.
• Electrically competent cells are prepared by
  growth in low NaCl conditions.
• Large scale preps are done, and aliquoted into
  smaller amounts for future use.
• Competent cells must kept on ice and stored at
  -80ºC.

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Heat-shock Transformation

• Thaw competent cells slowly on ice
• Add 200ng of plasmid DNA, or 20ul ligation mix,
  to 50ul cells (amount varies)
• Incubate on ice 20-30 min
• Incubate in a 42ºC water bath for 2 min to
  shock cells (open pores to allow DNA access)
• Place cells on ice for 2 min (closes pores and
  begins recovery)
• Add nutrient broth 1ml to cells
• Recover by shaking cells at 37ºC 1 hour to allow
  the production of antibiotic resistance proteins

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Electroporation

• Electroporation with high voltage makes
  biomembranes transiently permeable to allow the
  influx of DNA.

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Electroporation

• Thaw cells and chill cuvette on ice
• Add 200ng (1ul) of plasmid DNA to 50ul cells,
  transfer to cuvette, still on ice
• Set electroporator to 2.5kV, 25uF and 200-400
  ohms and put cuvette in place
• Pulse cells briefly (10 msec)
• Immediately add nutrient broth 1ml to cells
• Recover by shaking cells at 37ºC 1 hour to allow
  the production of antibiotic resistance proteins

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Plating Cells

• After recovery period, pellet cells briefly and
  resuspend in LB media
• Aliquot 10-100ul (amount varies based on
  transformation efficiency) of cell suspension
  onto LB plates that contain appropriate
  antibiotic
• Spread cells evenly using sterile glass or
  plastic spreader or glass beads
• Allow excess liquid to absorb at room temp 15
  min
• Incubate plates at 37C upside down for 16 hours
• The goal is to get independent colony formation

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Plating Cells
Glass rod
Agar plate with selective media
Formation of independent colonies
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Important Tips

• Do not freeze-thaw cells numerous times
• Use proper controls, supercoil DNA, and no
  insert controls for ligations
• Be sure plates are less than 1 month old, and
  were stored in the dark at 4ºC
• Warm plates in 37ºC incubator to dry agar
  surface
• Incubate plates upside down to avoid
  condensation drips
• Do not incubate plates longer than 16 h, or
  satellite colonies will form