Do you expect any colonies on the plates? Explain
MOLECULAR BIOLOGY ASSIGNMENT
I have attached a document containing the assignment. I ONLY need part 1 completed, NOT part 2. Part 1 is 2 questions each with 2 parts, so 4 questions. The assignment is designed to take approx. 1 hour.Thank you
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BIOS S12 -2011 PROBLEM SET #2 DUE DATE JULY 12 Part I is assigned to students registered for undergraduate and graduate credit whereas part II is only assigned to graduate students. Part I (25 points) Question 1 (12 points) The plasmid diagrammed below contains several unique restriction sites (E,X, and S), a restriction site present twice in the plasmid (A), a gene that confers resistance to ampicillin (AmpR), a gene that confers resistance to kanamycin (KanR) and an origin of replication (Ori). 1) The plasmid (3500 bp in length) is digested with EcoRI and XbaI. We assume that the loss of nucleotides located between the two restriction sites is negligible. a) A fragment of DNA (F, 500 bp in length) was obtained by digestion of genomic DNA with the restriction enzymes EcoRI and SpeI. The cleaved plasmid and cleaved fragment F are mixed together, and then treated with DNA ligase. Bacteria, unable to grow in the presence of kanamycin and ampicillin, were transformed with the ligation mixture. Transformed bacteria are plated either on dishes containing ampicillin or on dishes containing kanamycin. Do you expect any colonies on the plates? Explain (4 pts) b) In a different experiment, you wanted to create a modified version of the plasmid.
You digested the original plasmid with restriction enzyme A, purified the longer piece of DNA resulting from the digestion and treated the purified DNA with ligase. Then, bacteria unable to grow in the presence of kanamycin and ampicillin were transformed with the ligation mixture. Due to some technical problem, the purified piece of DNA is contaminated with copies of the original plasmid. Therefore the bacteria were transformed with a mixture of the original and the modified plasmid. Describe how you would quickly select bacteria transformed by the modified plasmid without extracting and sequencing the plasmid from transformed bacteria. (4 pts) 2) Assuming that you got bacterial colonies in the experiment described in part 1a. You…
BIOS S12 -2011 PROBLEM SET #2 DUE DATE JULY 12 Part I is assigned to students registered for undergraduate and graduate credit whereas part II is only assigned to graduate students. Part I (25 points) Question 1 (12 points) The plasmid diagrammed below contains several unique restriction sites (E,X, and S), a restriction site present twice in the plasmid (A), a gene that confers resistance to ampicillin (AmpR), a gene that confers resistance to kanamycin (KanR) and an origin of replication (Ori). 1) The plasmid (3500 bp in length) is digested with EcoRI and XbaI. We assume that the loss of nucleotides located between the two restriction sites is negligible. a) A fragment of DNA (F, 500 bp in length) was obtained by digestion of genomic DNA with the restriction enzymes EcoRI and SpeI. The cleaved plasmid and cleaved fragment F are mixed together, and then treated with DNA ligase. Bacteria, unable to grow in the presence of kanamycin and ampicillin, were transformed with the ligation mixture. Transformed bacteria are plated either on dishes containing ampicillin or on dishes containing kanamycin. Do you expect any colonies on the plates? Explain (4 pts) b) In a different experiment, you wanted to create a modified version of the plasmid.
You digested the original plasmid with restriction enzyme A, purified the longer piece of DNA resulting from the digestion and treated the purified DNA with ligase. Then, bacteria unable to grow in the presence of kanamycin and ampicillin were transformed with the ligation mixture. Due to some technical problem, the purified piece of DNA is contaminated with copies of the original plasmid. Therefore the bacteria were transformed with a mixture of the original and the modified plasmid. Describe how you would quickly select bacteria transformed by the modified plasmid without extracting and sequencing the plasmid from transformed bacteria. (4 pts) 2) Assuming that you got bacterial colonies in the experiment described in part 1a. You…
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