Data and Analysis
Medium
amp/no amp
Cells
- plasmid
1
- plasmid
2
+ plasmid
3
+ plasmid
4
Part II
No amp
+ plasmid
amp
Growth
Expected?
(yes or no)
Color
Expected
(blue/white)
Observed Growth
(no, lawn, #
colonies)
Observed
Color
(blue/white)
Reason for the
Result
amp
amp
No amp
With disrupted
B-galac gene
5
+ plasmid
amp
B-galac gene
not disrupted
6
If your observed results differed from your expected results, explain what you think may
have occurred in the space below…
1. Compare and contrast the number and/or color of colonies on each of the following pairs
of plates. What does each pair of results tell you about the experiment?
Plates to Compare
1&4
1&2
2&3
3&4
5&6
Result
Conclusion
2. What was the purpose of plating your + and – plasmid cells on the no amp plates? What
was the purpose of plating your + and – cells on the Amp plates?
3. In part I, what are you selecting for in this experiment? (i.e. What tells you that
transformation has been successful and the plasmid is in the recipient cells?)
4. Why did cells without the plasmid not grow on the amp plates?
5. Even though cells with the plasmid grew on amp plates, why did less grow than on the no
amp plate?
6. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per
g of plasmid DNA. The object is to determine the mass of the plasmid that was spread
on the experimental plate and was, therefore, responsible for the transformants observed.
Because transformation is limited to only those cells that are competent, increasing the
amount of plasmid does not necessarily increase the probability that a cell will be
transformed. A sample of competent cells is usually saturated with the addition of a
small amount of plasmid, and excess DNA may actually interfere with the transformation
process.
a. Determine the total mass (in g) of plasmid used. Remember, you used 10 l of
plasmid at a concentration of 0.005 g/l.
Total mass = volume x concentration
b. Calculate the total volume of cell suspension prepared.
c. Now calculate the fraction of the total cell suspension that was spread on the plate.
Volume suspension spread/total volume suspension = fraction spread
d. Determine the mass of plasmid in the cell suspension spread.
Total mass plasmid (a) x fraction spread (c) = mass plasmid DNA spread
e. Determine the number of colonies per g plasmid DNA. Express your answer in
scientific notation.
Colonies observed/mass plasmid spread (d) = transformation efficiency
7. What factors might influence transformation efficiency? Explain the effect of each
factor you mention. (You must have at least two)
8. Why might transformation be totally unsuccessful?
9. In part II, what does the blue vs. white colonies tell you?
10. So all of the white and blue colonies in part II have the plasmid? Why or why not?
11. You transformed E. Coli with pBlu plasmid. It contains genes for -galactosidase and
ampicillin resistance. Explain how you would insert a gene into this plasmid and explain how
you would use the ampr and -galactosidase genes to select clones that have your gene.
Between part I and II, you used this selection process, explain it. (list the steps how you wold
do it)