Download Scientific Experimentation

AP Biology
Transformation Lab Results
1. Label the E. coli cell above.
Ribosomes
Plasma membrane
LB (-)
LB/Amp (-)
LB =_________________
Plasmids
LB/Amp (+)
Name:
Period:
Chromosomal DNA
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 ________________
______________________________________________________________________
______________________________________________________________________
7. Which plates are meant to be control plates? ________________________________
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Explain your answers. ____________________________________________________
______________________________________________________________________
______________________________________________________________________
Explain the purpose of each step in 8 thru 10
8. The addition of transformation solution (CaCl2) the suspension___________________
_______________________________________________________________________
9. The addition of LB Broth to the suspension__________________________________
10. “Heat shocking” the E coli.______________________________________________
_______________________________________________________________________
11. The insertion site of the DNA molecule is called _____________________________
12. Enzymes that cut DNA at specific sites are called. ____________________________
13. The attraction in “Sticky Ends” are caused by _______________________________
13. Enzymes that catalyze phosphodiester bonds between two DNA fragments are
called________________________
Read: Appendix D - Gene Regulation online.
14. Explain the role of arabinose in gene regulation.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
15. Sketch the Arabinose Operon and GFP expression below.
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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)
(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: __________ =
Convert the answer above into scientific notation
transformants/ µg
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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