Q1.  You have a circular plasmid that has a single EcoRI site in it, as diagrammed in the figure below, which also shows the cleavage site for EcoRI. Choose the answer below that best represents what the end of the DNA molecule will look like once you cut the plasmid with EcoRI. Note that only the very ends of the DNA molecule are shown in the answers.

Q2. See the plasmid map below. Answer the questions

A. What sized fragments will be generated by cutting with EcoR1

B. Digestion with which enzyme combination would result in fragments of 1.6 and 1.8Kb

Q3. Figure shows the recognition sequences and sites of cleavage for the restriction enzymes SalI, XhoI, PstI, and SmaI, and also a plasmid with the sites of cleavage for these enzymes marked.

A. After which of the five treatments listed below can the plasmid re-form into a circle simply by treating it with DNA ligase? Assume that after treatment any small pieces of DNA are removed, and it is the larger portion of plasmid that will reassemble into a circle.

After digestion with __________.

1. SalI alone

2. SalI and XhoI

3. SalI and PstI 

4. SalI and SmaI

5. SmaI and PstI

B. Let's suppose you have a cloning vector that only has one restriction site available for cloning , Sma1.  You must clone a DNA fragment into it that has XhoI sites on its ends. Assuming you have access to all DNA modifying and synthesizing enzymes found in E. Coli cells and any reagents you need. Can you think of a way to clone your fragment into the vector? 

Q4. You have a piece of circular DNA that can be cut by the restriction nucleases EcoRI, HindIII, and NotI, as indicated in the Figure below.

Label the following statements true or false.

(a) One piece of DNA will be obtained when this DNA is cut by NotI.

(b) A piece of DNA that cannot be cut by EcoRI will be obtained by cutting this DNA with both NotI and HindIII.

(c) Two DNA fragments that cannot be cut by HindIII will be obtained when this DNA is cut by EcoRI and NotI.

(d) Two DNA fragments of unequal size will be created when this DNA is cut by both HindIII and EcoRI.

Q5. Name three features that a cloning vector for use in bacteria must contain. Explain your answers.

Q6. What additionally would be required for a plasmid to be used as an expression vector?

At the beginning of the class I mentioned that we would incorporate exercises in ethics in science.  Next class we will do this in place of in-class questions. An important part of dealing with ethical problems is to identify the potential ethical problem and identify stakeholders and arrive at potential solutions. 

Title: Consequences of ethical violations by scientists and conducting science "in the media".

Earlier this year there were multiple outbreaks of measles and mumps- remember Disneyland and the NHL?  Immunization rates are dropping and parents are withholding vaccinations from their children due to a fear of them developing autism. This belief originated from from a single highly publicized 1998 paper that appeared in a medical journal. The doctor who authored the publication made claims at a press conference that far exceeded those allowable by the journal and the peer review process. Further, it was reported that some of the parents of the children in the study were recruited by a lawyer preparing a lawsuit against vaccine manufacturers and that the doctor subjected patients to procedures. It was also discovered that the doctor had an agreement with this law group, which he did not disclose. Finally, claims in the original paper were later found to be false, or fabricated. The original paper has been retracted and the findings dismissed by the medical community. Despite this, advocacy groups continue to fight against immunizations and some parents do not vaccinate their children citing the risk of autism.

Please consider the following and answer the questions:

1. What are the perceived ethical violations in this case?

2. What are facts?

3. What are the implications of the violations? Short term? Long term?

4. Who is harmed?

5. What were better options?

6. What future steps should be taken?