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AP Lab 6

Completing the Research Notebook for AP Biology Lab #6.....Molecular Biology

Resource: Lab Six, Molecular Biology

Pages 64-77 in the AP Biology Lab Manual

 Note: you will be using handouts from Carolina Biological kits for this laboratory procedure.

 

Part 1: Title

Develop a title in the form of a question after completing the pre-lab.

 

Part 2: Objectives (What are the objectives for this laboratory?)

 

Part 3: Prelab Questions

Transformation

1. Describe E. coli and its singular circular chromosome.

2. Explain plasmids, including “R” plasmids.

3. Explain 3 ways that genes can be transferred between bacteria.

4. Explain what is meant by “competent”.

5. Explain figure 6.1

6. Figure 8 is the plasmid we will be using to transform. Inside the circular plasmid there are two bold, thick black

semicircles. These are the two genes on this plasmid that we are interested in. What are they?

7. What is “X gal” and what will happen if bacteria are transformed with this plasmid?

Gel Electrophoresis

8. What are restriction endonucleases (enzymes)?

9. Explain how they are named (EcoRI)???

10. Explain Figure 6.2b (page 69, AP Biology Lab Manual)

11. In this experiment, how will the three samples of DNA be cut?

12. Explain what causes the DNA to “move” through the agar gel in the gel chamber and what causes the

separation of the bands into “DNA fingerprints”.

IT IS VERY IMPORTANT THAT YOU READ THESE PAGES AT LEAST TWICE.

THE PROCEDURE IS VERY COMPLICATED, AND YOU NEED TO BE PREPARED.

TRANSFORMATION: READ PAGES 4 AND 5 (procedure from Carolina Biological kit)

ELECTROPHORESIS: READ PAGES 10, 11, 12 (procedure from Carolina Biological kit)

 

Part 4: Method

Transformation: Starter Plates of E. coli will be obtained which have a source of bacterial cells in an exponential

growth phase. The plasmid used is pBLU which has a gene for ampicillin resistance and a gene which will cause a

chemical called X gal to form blue colonies. In a controlled experiment, the E. coli bacteria cells will be made

“competent” by suspending them in cold calcium chloride, incubating them on ice, adding the plasmid, heat shocking

them, and then subjecting them to ice. The cells will be spread on luria broth agar with and without ampicillin for 24

hours in an incubator.

Gel Electrophoresis: A gel casting tray will be sealed on each end with masking tape, liquid agar will be poured into

the tray and allowed to solidify. The tray will be placed in a gel box so that the comb is on the “negative” or black

end, and TBE buffer will be poured over the agar. The comb will be removed. The box will be covered with wrap.

The next morning, micropipets will be used to transfer DNA subjected to various restriction enzymes into the wells,

and the power source will be turned on.

 

Part 5: Data

Design data tables that will enable you to collect qualitative and quantitative data from both procedures:

transformation and gel electrophoresis of DNA.

 

Post-lab:

Part 6: Questions

The questions will be found on student guides from the Carolina Biological kits for these labs.

Page 6 and 7 #1-10

Page 12 and 13 # 1-4

 

Part 7: Graph

You will need to construct a graph in order to determine the number of base pairs for the fragments with the EcoRI

restriction enzyme cut in the DNA gel electrophoresis lab.

The graph paper used is semilog graph paper. Directions will be reviewed during class. The directions are on the

student guide for the lab. The directions are also on page 72-73 of the AP Biology Lab Manual.

 

Part 8: Theme Correlation

Explain how this lab demonstrated both inquiry and structure fits function.

Inquiry: How was the transformation experiment controlled? Was biotechnology demonstrated?

Structure Fits Function: How can genes of interest be placed into plasmids?

 

Part 9: Conclusion

1. Use the transformation lab results to explain how plasmids can be used in genetic engineering.

2, Gel electrophoresis: Use the lab results to explain how restriction enzymes work; the function of an electrical

field during gel electrophoresis; the function of the agarose gel.

3. What is a restriction site? How could a mutation that alters a restriction site be detected by gel electrophoresis?

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