Bio201l lab 7 Plasmid Digest Activity Sheet
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Lab 7 In lab Digest activity
Bio201L Lab 7 Plasmid Digest Activity Sheet Imagine you have a recombinant plasmid with a single 2.5 kb Eco R1 yeast fragment insert. You have determined that the insert does not have a Bam H1 recognition site.
A. In the space at right draw a map of this recombinant plasmid indicating the insert, the vector, their sizes, along with the Eco R1 and Bam H1 sites.
B. Using the gel image below, draw in the expected band sizes for the recombinant plasmid if it was digested with Eco R1 (Lane 1) and digested with Bam H1 (Lane 2).
C. Now, imagine that you have 100 copies of the recombinant plasmid in a test tube with the Eco R1 enzyme and, instead of letting the Eco R1 digest for the full 45 minutes, you stop the digest after 20 minutes, which means that not all of the Eco R1 sites on all of the plasmids are cut: 75% of the recombinant plasmids were cut at both Eco R1 sites on the plasmid, and 25% of the recombinant plasmids were cut at only one of the two sites (i.e., they were partially digested). Using the gel at right (Lane 3), draw in the expected Eco R1 band sizes you would expect to see from the 20-minute digest of the plasmids. Compare the results of Lane 3 with Lane 2. What do you notice?
D. Assuming that you started with 100 recombinant DNA plasmids in your test tube, how many DNA fragments would you expect in each band you have drawn in lane 3?
Which of the bands in lane 3 would you expect to have the greatest fluorescent intensity? How did you decide this?
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