Exercise 6B: Restriction Enzyme Cleavage of DNA and Electrophoresis
Analysis Questions:
1. Discuss each of the following factors and their influence on the gel electrophoresis
results:
a)
b)
c)
d)
Voltage used
Running time
Amount of DNA
Reversal of polarity
2. Two small restriction fragments of nearly the same base-pair size appear as a single
band, even when the sample is run to the very end of the gel.
What could be dome to resolve the fragments? Why would it work?
3. What is a plasmid? How are plasmids used in genetic engineering?
4. What are restriction enzymes? How do they work? What are recognition sites?
5. What is the source of restriction enzymes? What is their function in nature?
6. Describe the function of electricity and the agarose gel in electrophoresis.
7. If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000,
2500, 2000, and 400 base pairs, sketch the resulting separation by electrophoresis.
Show starting point, positive and negative electrodes, and the resulting bands.
8. What are the functions of the loading dye in electrophoresis? How can DNA be
prepared for visualization?
9. Use the graph prepared from the lab data to predict how far (in mm) a fragment of
8000 base pairs would migrate.
10. How can a mutation that alters a recognition site be detected by gel electrophoresis?
11. Error Analysis: What are some possible errors that could have occurred during the
lab that would affect results? (Hint: Think about lab technique, handling multiple
samples, interpretation, etc.)
12. Conclusion: How does gel electrophoresis separate DNA fragments? Can you use
a standard curve to determine the size of unknown DNA fragments?
13. Can this procedure be useful in determining whether or not a plasmid was
successfully incorporated into a host bacterial genome? Explain using the data.
14. How can this technology (gel electrophoresis procedure) be beneficial for society?