Analyze your gel photograph and discuss the following questions with your partne
ID: 63336 • Letter: A
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. Why is it important to verify that you have the correct recombinant plasmid?
The name itself says that it is recombinant. A gene of interest is cloned into a plasmid. It is certain to know the gene is inserted into a plasmid.
2. How did your actual gel results compare to your gel predictions?
Always gels can’t give the best result even though the sample is dyed.
3. Do you see any bands that are not expected? What could explain the origin of these unexpected bands?
If any other bands are therein the gel, might be the sample is contaminated.
4. Does the gel show that your restriction digest and ligation procedures were successful? Describe the evidence you used to make this assessment.
It is important to load a primer for the gene which is cloned. It will help in assessing the results.