pGLOTM Bacterial Transformation
Extension Activity:
Calculation of Transformation
Efficiency
Bioluminescence
of Aequorea
victoria
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)
Green fluorescent cell count
 Observe LB/amp/ara plate under UV light and
count the number of colonies on the plate
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
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
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
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
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
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)
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!!!
Factors Affecting Transformation Efficiency
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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
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.