Analyze your gel photograph and discuss the following questions with your partne

Question

Analyze your gel photograph and discuss the following questions with your partner. Be prepared to share your answers with the class.

1. Why is it important to verify that you have the correct recombinant plasmid?

2. How did your actual gel results compare to your gel predictions?

3. Do you see any bands that are not expected? What could explain the origin of these unexpected bands?

4. Does the gel show that your restriction digest and ligation procedures were successful? Describe the evidence you used to make this assessment.

5. In the geK– and geA– lanes, do you see evidence of multiple configurations of plasmids? Explain your answer.

6. In the geK+ and geA+ lanes, do you see evidence of complete digestion? Explain your answer.

7. In which lane would you expect to find the rfp gene and the ampR gene in the gel photograph? Are you able to locate these two genes? Explain your answer.

8. Compare the lanes that have linear fragments with the lanes that have plasmids. Is there a difference in the shape of the bands between these two DNA forms?

9. In Laboratory 3, you described all the possible plasmids that you could make by ligating the digesting fragments of the pKAN-R and the pARA plasmids. Two of the rfp gene fragments (807 bp each) may form a circularized fragment because each end of the fragments terminates in BamHI and HindIII sticky ends. Is there evidence of a circularized 1,614 bp fragment in the geLIG tube lane? Explain your answer.

Explanation / Answer

1. During the insertion process, the plasmids may anneal to their complementary ends and form a closed circle even without an insert, or the insert may not be in proper iorientation. Screening is done to eliminate such plasmids and to select only those plasmids with foreign gene insert in proper orientation

3. The unexpected bands are those of fragments that did not insert into plasmids, or plasmids without a foreign gene insert, or both

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.