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pGLO TM Bacterial Transformation

pGLO TM Bacterial Transformation. Extension Activity: Calculation of Transformation Efficiency. Bioluminescence of Aequorea victoria. Transformation Efficiency.

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pGLO TM Bacterial Transformation

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  1. pGLOTM Bacterial Transformation Extension Activity: Calculation of Transformation Efficiency Bioluminescenceof 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 (128-1120 transformed colonies)

  3. Green fluorescent cell count • Observe LB/amp/ara plate under UV light and count the number of colonies on the plate

  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

  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.

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