Analyze the fictitious plasmid pltw shown below

Activity 6.1.1 Restriction Enzyme Challenge

Equipment

● Laboratory journal
● Highlighter (optional)

Some restriction enzymes (e.g., EcoRI) cut DNA unevenly, leaving jagged edges called sticky ends. These pieces can snap together like the pieces of a puzzle. The process of DNA ligation “links” two or more of these pieces together to create unique DNA sequences.

● How many fragments would result if the DNA above was digested with EcoRI? List the size of each resulting fragment.

there will be four fragments they will be 300, 700, 3500, 300

● You experienced a pipetting mishap in the lab. You are not sure that your restriction enzyme mixture made it into your sample. Explain what you would see on a gel if restriction enzymes were not added and digestion was unsuccessful.

There would be one band that would be 4350 base pairs long

fragments as well as the size of each fragment.

o Digestion with EcoRI

1; 4500

o Digestion with EcoRI/PstI

fragment of DNA shown to the right into the plasmid vector. Think about how to open the circle

so that you create sticky ends compatible with those of the fragment.

6. View the new version of the pLTW plasmid shown below. Your goal is to remove the 1500bp fragment inserted in Step 4 and replace it with the new fragment shown to the side of the plasmid. Digest the starting plasmid with HindIII, add the new fragment to the mixture, and ligate using the enzyme ligase. NOTE: This new fragment of DNA contains an extra restriction site.

possible products. Which enzyme would help you best distinguish between the five possible results? Think about the number of fragments that would be produced and their potential size. Assign a lane for each possible product on the gel diagram below and draw the results of your digestion.

7. Use the information below to assemble a plasmid map. Show the location of restriction sites

and label the distances between these sites in base pairs.

o DNA digested with PstI and HindIII – 500bp, 300bp, 200bp fragments

Conclusion

3. You are trying to match DNA found at a crime scene with two possible suspects. Why would it be a better use of your time and resources to analyze the DNA using a double restriction digest rather than a single digest? HINT: Think back to your DNA Detectives gel analysis in HBS.