Plasmid Mapping Worksheet

Plasmid Mapping: Restriction Enzyme Analysis

Read each question carefully and provide detailed answers. Show all calculations where applicable.

1. Which of the following is the primary purpose of plasmid mapping?

2. Which technique is commonly used to separate DNA fragments after restriction enzyme digestion?

3. Restriction enzymes recognize specific nucleotide sequences called   sites.

4. The fragments generated by restriction enzyme digestion can be visualized by staining with   and exposing to UV light.

5. A circular plasmid of 5000 base pairs (bp) is digested with a single restriction enzyme, EcoRI, and yields a single fragment of 5000 bp. If the same plasmid is then digested with two restriction enzymes, EcoRI and BamHI, and yields two fragments of 2000 bp and 3000 bp, what can you conclude about the location of the BamHI site relative to the EcoRI site?

6. You have a circular plasmid of unknown size. You perform restriction digests with three different enzymes (A, B, and C) and analyze the fragments by gel electrophoresis. The results are as follows:

- Digestion with Enzyme A alone: Fragments of 6000 bp and 4000 bp.

- Digestion with Enzyme B alone: Fragments of 7000 bp and 3000 bp.

- Digestion with Enzyme C alone: Fragments of 5000 bp and 5000 bp.

- Double digestion with Enzymes A and B: Fragments of 2000 bp, 4000 bp, and 4000 bp.

- Double digestion with Enzymes B and C: Fragments of 1000 bp, 3000 bp, and 6000 bp.

- Double digestion with Enzymes A and C: Fragments of 1000 bp, 4000 bp, and 5000 bp.

a) What is the total size of the plasmid?

b) How many recognition sites does each enzyme have on the plasmid?

c) Draw a circular restriction map of the plasmid, indicating the positions of the restriction sites for enzymes A, B, and C. Label the distances between sites in base pairs.