BIOL327 Molecular Biology Laboratory Schedule Fall 2000
Instructor:Marie Pizzorno Department of Biology Bucknell University Office: BB205 Phone: (570) 577-3084 pizzorno@bucknell.edu	Wed 2-5pm BB219

Laboratory:

This lab will be run as a semester long experiment. There will be one report due on the last day of class (late reports will not be accepted), which should be written in the form of a scientific paper. I will give you more details on this later. In addition, at the end of the semester each lab group will give a presentation on their data or on a relevant scientific article.

The goal of the experiment will be to "shotgun" clone the genomic DNA of an organism into a plasmid vector and obtain the sequence of one of the inserted fragments of DNA. First you will isolate a large quantity of the plasmid vector, pGEM7z, and purify it by cesium chloride density centrifugation. Then you will isolate genomic DNA from whole bacterial or eukaryotic cells, digest it into millions of fragments with a restriction endonuclease, and insert those fragments into the prepared vector. The next step is to isolate some of the resulting colonies of bacteria and purify plasmid DNA from them. Finally you will perform a restriction mapping of the recombinant plasmid and sequence the inserted genomic DNA. At the end of the semester you should be able to identify what gene your randomly inserted piece of DNA came from and give me some basic analysis of this sequence.

I will make available several organisms that you may use as your source of genomic DNA. These will include bacteria and mammalian tissue culture cells from several species. Instead you may choose to use something different. Some possible choices are bovine DNA (from beef heart or liver), rat DNA (liver), insect or plant DNA. You should be warned that tissues can be more difficult to purify DNA from than the cells I have chosen, though I can help you locate the best protocol to use with these other sources.

Each week's activity will be described and demonstrated during the pre-lab discussion. Please arrive to lab on time so that you do not miss any vital information. Because the actual procedures are difficult to accomplish in a 3-hour time period, you will have to do some work outside of the scheduled lab time, though if you come to lab having read and familiarized yourself with the handout, you will be better able to accomplish everything in the allotted time frame. How much work you need to perform outside of lab depends on how efficient and organized you are during lab.

You should keep a detailed and organized lab notebook. I don't care what kind of notebook you use, though a single subject spiral bound notebook is probably the easiest. These will be collected before Fall Break and graded for completeness and accuracy. The data and procedures you record will be vital when you write-up the lab report at the end of the semester. In addition, it will be hard to determine where problems occur in the procedure (and for me to help you fix those problems) if you havenŐt written down exactly what you did in lab.

Lab Rules/Safety:

1. Call x1111 if there is an emergency!!
2. Absolutely No Eating or Drinking in the lab at any time.
3. Be careful with acids/bases and phenol as they can burn your skin. Wash acids/bases with plenty of H2O. Phenol is best removed with PEG.
4. Be aware of safety features in the lab (extinguisher, eye wash, spill kit).
5. Discard waste in the appropriate containers; hazardous liquids, biomedical, etc. Do not discard paper towels and other "non-contaminated" waste into the red boxes. Please reserve these for actual biomedical waste (tubes, tips, plates) and use the regular trash cans for other things.
6. Turn off all equipment when you leave the lab, with the exception of water baths. Especially important, make sure the lamps are off on the spectrophotometers if you have used them.
7. Please make sure you understand how to operate a piece of equipment before you use it. If you have any questions please ask me.
8. Be courteous! If you use the last of something, replace it or notify me ASAP. Also keep the lab clean and wipe up any liquid spills, especially on the floor.
9. Make sure you properly label all of your solutions and tubes. The more information on the sample, the better the chances you will not get them mixed up or confused. Place all of you samples in the appropriately labeled boxes at the correct temperature (fridge = 4oC, freezer = -20oC) at the end of each lab period.