1. pGLOTM Bacterial Transformation
Extension Activity:
Calculation of Transformation Efficiency
Bioluminescence of Aequorea victoria

2. 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 ( transformed colonies)

3. Green fluorescent cell count
Observe LB/amp/ara plate under UV light and count the number of colonies on the plate
Each colony can be assumed to be derived from a single cell. As indiv. Cell reprod. More and more cells are formed and dev. into what is termed a colony.

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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)

10. 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!!!

11. 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
See full notes for further description

12. 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.