pGLOTM Bacterial Transformation

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pGLOTM Bacterial Transformation
Extension Activity Calculation of
Transformation Efficiency
Bioluminescence of Aequorea victoria
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Transformation Efficiency

• Transformation efficiency is a quantitative value
  that describes how effective you were at getting
  a plasmid into bacteria. The number represents
  the number of transformed colonies produced per
  microgram of DNA added.
• This protocol has been determined to have a
  transformation efficiency between 8.0 x 102 and
  7.0 x 103 (128-1120 transformed colonies)

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Green fluorescent cell count

• Observe LB/amp/ara plate under UV light and count
  the number of colonies on the plate

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Determine Amount of pGlo plasmid DNA in bacterial
cells

• How much pGlo DNA was in the bacterial cells
  spread on the LB/amp/ara plate?
• Total amount of DNA at beginning of experiment
• Fraction of DNA (in the bacteria) actually spread
  onto the LB/amp/ara

• pGlo DNA spread (µg) DNA used (µg) X fraction
  of DNA

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Determining the total amount of DNA

• DNA (µg) (conc. of DNA (µg/µl) X (vol. of DNA
  (µl))
• 10 µl of DNA at a concentration of 0.08 µg/µl was
  used in this experiment
• DNA (µg) 0.08 µg/µl X 10 µl
• DNA (µg) 0.8 µg
• This number will be multiplied by the fraction of
  DNA used to determine the total amount of DNA
  spread on the agar plates

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Determine the fraction of pGlo plasmid DNA spread
onto the LB/amp/ara plate

• Fraction of DNA used Volume spread on LB/amp
  plate
• Total volume in test tube
• Look in the laboratory procedure and locate all
  the steps where you added liquid to the reaction
  tube and add the volumes
• Fraction of DNA used 100 µl
• 510 µl
• Fraction of DNA used .2

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How much DNA was spread on the LB/amp/ara plate?

• pGlo DNA spread (µg)DNA used (µg) X fraction of
  DNA
• pGlo DNA spread (µg) 0.8 µg X .2
• pGlo DNA spread (µg) .16

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Determine Transformation Efficiency

• Transformation Efficiency Total of cells on
  agar plate
• Amount of DNA spread on agar plate
• Transformation Efficiency (transformants/µg)
• Use scientific notation to report transformation
  efficiency

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Additional Calculations

• Determine transformant colony count from
  transformation efficiency
• From a known transformation efficiency, how many
  transformant colonies would be expected to grow
  on the LB/amp/ara plate?
• Transformation Efficiency Total of cells on
  agar plate
• Amount of DNA spread on agar plate
• of Cells on Agar Plate (Trans Eff) X (Amount
  of DNA)

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Additional Analysis

• How many individual transformed bacterial cells
  were on their plates at the time of plating?
  Were they visible?
• Same number of individual cells as there now are
  COLONIES.
• Colonies 24hrs X 60min/hr X 1 doubling/40min
  36 doublings
• of Cells/colony (start with one cell) 236
  6.8 x 1010
• Almost 70 billion cells per colony!!!

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Factors Affecting Transformation Efficiency

• Refrigeration of cultured E.coli plates
• Size of colony originally suspended in the CaCl2
• Amount of plasmid added
• Time on ice allowing plasmid to come in contact
  with cells
• Heat Shock Treatment
• Diligence in going directly from ice to 42oC back
  to ice
• Amount of LB broth added
• Recovery time in LB broth
• Amount of cells spread on plates
• Spreading transformants and controls

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Group Comparison

• Students compare results and review all of the
  steps that are variable. The students see how
  technique and protocol are key to obtaining
  results.
• Some classes will obtain 100 transformation
  results (glowing bacteria), but 75 to 80
  successful transformation is acceptable for
  student groups.