AP Biology
Transformation Lab Results
Name_____________________________
Date ______________________ Per ___
1. Label the following on E. coli cell below: Ribosomes, Plasma membrane, Plasmids, Chromosomal DNA
Observe the results on the plates under normal lighting conditions. Then observe using a UV light.
Draw your results from the plates on the corresponding spaces below.
I
LB (-)
LB =_________________
II
LB/Amp (-)
III
IV
LB/Amp (+)
LB/Amp/Ara (+)
Amp = _________________ Ara =_________________
(+) =_________________
(-) =_________________
2. Which plate(s) should have exhibited bacterial growth? ________________________
Explain: __________________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
3. Which plate shouldn’t have contained any bacterial growth? ____________________
Explain: __________________________________________________________________________
___________________________________________________________________________________
4. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic
ampicillin, then what might be inferred about the other genes on the plasmid used in the experiment? ___________________________________________________________________________________
___________________________________________________________________________________
5. Which plate shows bioluminescence when exposed to UV-light? ________________
6. Explain why the plasmid does not fluoresce, but the E. coli does _____________________________
___________________________________________________________________________________
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7. Which plates are meant to be control plates? ________________________________
Explain your answers. _________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
8. The insertion site of the DNA molecule is called _____________________________
9. Enzymes that cut DNA at specific sites are called. ____________________________
10. The attraction in “Sticky Ends” are caused by _______________________________
11. Enzymes that catalyze phosphodiester bonds between two DNA fragments are called ____________
Explain the purpose of each step in #12-14:
8. The addition of transformation solution (CaCl2) the suspension
9. The addition of LB Broth to the suspension
10. “Heat shocking” the E coli.
Read: Appendix D - Gene Regulation online to help answer #15-18.
15. Describe the arabinose operon.
16. Sketch the Arabinose Operon.
17. Explain the role of arabinose in gene regulation.
18. Explain why the GFP gene is only expressed on the plate with arabinose.
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Transformation Efficiency
Count the total # of cells growing on the LB/Amp/Ara plate:
To determine the amt of DNA (pGLO) in the bacterial suspension spread on the LB/Amp/Ara plate
follow the steps below.
1. Calculate the total amt of DNA (µg) used in the experiment
Amt of DNA used = (concentration of DNA µg/µl) x (volume of DNA µl)
(You used 10 µl of pGLO at 0.03 µg/µl concentration, meaning each microliter of solution contained
0.03 µg of pGLO DNA)
Amt of DNA used = (
µg/µl) x (
µl) =
____µg DNA
2. From the lab directions determine the fraction of DNA in the bacterial suspension spread on
LB/AMP/ARA plate:
Fraction of DNA used in exp = Volume spread on LB/Amp/Ara plate
Total sample volume in test tube
Fraction of DNA spread:
__
µl =
µl
3. Multiply the total amt of DNA used x the fraction of DNA spread = DNA (pGLO) in the bacterial
suspension spread on the LB/AMP/ARA
_____ µg DNA x _____ =
_____ µg DNA in spread
__
Transformation efficiency: Total # of cells growing on the LB/Amp/Ara plate
Amt of DNA in the E. coli suspension spread on the plate
Transformation efficiency: __________ =
transformants/ µg
Convert the answer above into scientific notation
transformants/ µg
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Transformation Efficiency Sample Problems.
Problem 1
Calculate the transformation efficiency using the following experimental results.
DNA Plasmid concentration: 0.03 µg/µl
250 µl transformation solution (CaCl2)
10 µl of plasmid solution
250 µl LB broth
100 µl cell suspension spread on the plate
227 colonies counted
transformants/ µg
Transformation efficiency: __________ =
transformants/ µg
Convert the answer above into scientific notation
Problem 2
Using the same concentration of DNA, and fraction of cells spread on the plate, plus a transformation
efficiency of 3 x 103 bacteria/ µg of DNA, calculate the number of transformants that would be expected
to grow on the LB/Amp/Ara plate.
Total cells =
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