Notes to Class

Lab 12: Restriction Mapping of pBR322

During today's wet laboratory on restriction analysis of the plasmid pBR322 you will map the locations of enzymes EcoRI, HincII and PvuII. In addition to performing this restriction analysis, you are going to map an additional 2 restriction enzymes onto your pBR322 map. To do this, you will use a virtual enzyme cutting site at New England Biolabs (NEB). 

Directions:

1. At the NEB site, you should click on NEBcutter. This will launch a program called NEBcutter V1.0.

2. You will see that pBR322 is one of the test sequences already in the NEBcutter database. (This program can download any DNA sequence and subject it to virtual digestion).

3. Click on the link to pBR322. This will give you a circular map of the plasmid with cutting sites shown for restriction enzymes that cut one time in the plasmid.

4. You are going to perform custom digests to the plasmid DNA using 2 additional restriction enzymes. The enzymes that you should use are BseVI and BsaI.

5. You will need to decide which digests to perform to figure out where these enzymes map in relation to the enzymes that you physically mapped in class. (e.g. you may need to do digests with these enzymes and your original 3 from class).

6. In your notebook, we want to see a sketch or print out of the digests that you performed, we will also want to see a discussion of how you figured out the map locations of the enzymes and finally, provide us with a circular map of pBR322 that shows all restriction sites and open reading frames (genes).

7. Obviously, you could just download the entire pBR322 map to get this information. However, if you do this, you will not get credit for the lab. You must determine the map locations via enzyme digestion. You can, however, check your class digestion data against the virtual digestion data.