Transformation Lab using pGLO
Analysis Questions:
These questions (in bold) are to be copied and then answered in complete sentences in your lab book or for your
formal lab report. (you do not need to copy the hints in italics)
1. What is the purpose of the plate marked –pGLO LB? Explain fully.
2. If the genetically transformed cells can live in the presence of the antibiotic ampicillin, then what might be
inferred about the other genes on the plasmid called pGLO?
3. How do you know that you actually transformed any bacteria?
(hint: Which petri dish is especially useful in making this determination? Explain.)
4.
(hint/comment: Very often an organism’s traits are caused by a combination of its genes and its
environment. Think about the green color you saw in the genetically transformed bacteria)
What two factors must be present in the bacteria’s environment for you to see the green color?
(another hint: one factor is in the plate and the other factor is in how you look at the bacteria)
5.
Calculate Transformation Efficiency
You are about to calculate the transformation efficiency, which gives you an indication of how effective you
were in getting DNA molecules into bacterial cells. Transformation efficiency is a number. It represents the
total number of bacterial cells that express the green protein, divided by the amount of DNA used in the
experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The
transformation efficiency is calculated using the following formula:
Transformation Efficiency=
Total number of colonies growing on the agar plate
Amount of DNA spread on the agar plate (in ug)
To do this - determine the number of green fluorescent colonies growing on your LB/amp/ara plate and the
total amount of pGLO (plasmid DNA) in the bacterial cells spread on the LB/amp/ara plate.
You put 100 ul of the bacteria solution on your petri dish.
Therefore the amount of DNA spread 100 ul/510ul = .196
Use .196 x 0.08 ug/ul (concentration of pGLO in 10ul)
Answer is 1.57 x 10 -2 ug ( of DNA spread) Use that number to determine your efficiency!