Biol 297C - Cell & Molecular Biology Lab

For the 2nd half of the course you will be introduced to several important molecular biology techniques, including:

• restriction enzyme digestions,
• agarose gel electrophoresis,
• plasmid DNA-mediated bacterial transformation,
• DNA purification,
• DNA sequence analysis, and
• the polymerase chain reaction.

These techniques will enable you to become familiarized with the manipulation of cloned DNA constructs. The overall goal is to subclone a fragment of yeast genomic DNA and obtain DNA sequence data. This information will then be used in DNA sequence database searches to identify which region of the yeast genome you have isolated. You will then determine what is known about the specific yeast gene or genomic region which you have identified.

While there will be no exams in this section of the course, you will be evaluated by your performance in 3 areas -

1. class participation,
2. lab write-up, and
3. lab meeting.

Class participation basically means attending each lab and being actively involved in performing the experiments and discussing issues and questions that may arise. The lab write-up will be due at the end of class and will cover one particular experiment (to be assigned later) as well as your overall cloning and sequence results. Finally, we will have a lab meeting at the end of class where each group will have 15 minutes to present the material in their lab write-ups to the rest of the class.

Note: we will discuss the format of the lab write-up and lab meeting in more detail later in the course.

joe@bio.umass.edu

Morrill I room N426 (5-0468)

Feel free to contact me regarding any questions you may have on the laboratory experiments or other aspects of the course.

Week 1 (March 29/30) - Digest yeast genomic DNA and pBluescript plasmid vector DNA with Eco RI and Bam HI restriction enzymes. Run out small amounts of the samples on an agarose gel to verify complete digestion. Freeze remainder of samples until next week.

Week 2 (April 5/6) - Run out rest of digests on an agarose gel. Cut out the appropriate bands and isolate DNA via spin columns and ethanol precipitation. Run out a small amount of the purified DNAs on an agarose gel to check recovery. Set up DNA ligations and let incubate overnight.

Week 3 (April 12/13) - Pour LB/Ampicillin plates. Transform DNA ligations into competent E. coli bacteria. Plate transformed bacteria onto LB/Ampicillin plates. (Visit Biology Department DNA Sequencing Facility?)

Week 4 (April 19/20) - NO CLASS -PATRIOTS DAY HOLIDAY

Week 5 (April 26/27) - Perform DNA mini-preps on Ampicillin-resistant bacterial colonies. Digest DNAs with Eco RI and Bam HI and check for yeast DNA inserts via agarose gel electrophoresis. Submit DNAs for sequence analysis.

Week 6 (May 3/4) - Perform polymerase chain reaction (PCR) assays on mini-prep DNAs using pBluescript primers. Analyze PCR products via agarose gel electrophoresis. Analyze DNA sequence data via BLAST searches of yeast genome database.

Week 7 (May 10/11) - Turn in lab write-ups and have lab meeting to discuss experimental results.