Restriction Enzyme AP Lab Write-up Guidelines
Due Fri, 1/17 or Thurs, 1/16 by 3:30 for +5
Title
I. Introduction
 General Overview
 Purpose/Explanation of Lab
II. Materials & Methods
 Summary of procedure followed
 “For a complete listing . . . “
III. Results
 Data Table
o Sample Data Table found on Lab Protocol WS posted
 Graph
o I will provide you with a copy of semi-log paper for graph construction for your lab write-up
o After plotting DNA standard points, draw a line of best fit to use for interpolating fragment sizes for Child 1 and Child 2.
IV. Questions – Please answer questions in complete sentences.
1. Explain the importance and value of restriction enzymes in DNA testing.
2. What are two purposes of the loading dye used in gel electrophoresis?
3. Compare the DNA fingerprints produced by restriction digestion of the PCR products generated from the children’s DNA and the
DNA fingerprints created by digestion of the PCR products from the control CF mutant and wild-type DNA. Which pattern does the
DNA fingerprint from each child’s DNA match – the pattern from the mutant DNA, the pattern from the wild-type DNA, or a
combination of both? Explain (provide data to support your answer).
4. Referring to question #3, what does this tell you about the genotype of each of the children with respect to this CF mutation?
5. Use your graph to predict the distance a fragment of 6000 bp would migrate.
6. Remember that this particular mutation in the cystic fibrosis gene destroys a restriction enzyme site. If you did not know about the
destroyed restriction site, what about the fragment patterns on your gel would indicate to you that a restriction enzyme had been
destroyed?
7. A certain restriction enzyme digest results in DNA fragments of the following sizes: 4400 base pairs, 2200 base pairs, 1600 base
pairs, and 600 base pairs. Make a sketch of a gel showing the resulting separation by electrophoresis. Indicate the starting point,
positive, and negative electrodes.
8. Explain the principle of “sticky ends”. Why are they useful in molecular genetics?
A
B
9.
A represents a linear chromosome, B is a circular bacterial chromosome.
a. If you were to digest both strands (A and B) with EcoRI, how many DNA fragments would result for each? Explain.
b. What enzyme(s) would you use to cut DNA A, keeping the “Red” gene intact?
10. Given the restriction sequences shown above,
a. In order to insert the “Red” gene into the bacterial chromosome and use the “Blue” gene as a loss of function reporter
gene, which restriction enzyme would you use? Explain.
b. Could HindIII be used? Explain.
11. Examine the DNA electrophoresis results shown below. Is it possible that the individual tested is the father of the child? Explain
your answer.
*No Conclusion or Error Analysis Required for this Lab Write-up! *
Academic Pledge