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This class is primarily a laboratory techniques class. There will be lectures associated with some of the labs, particularly during the first half of the semester (see annotated syllabus below). The lectures are intended to give you the theoretical background needed to understand the rationale behind the laboratory experiments. The laboratory experiments are interrelated and sequential. The results of one laboratory experiment are your starting materials for the next set of experiments.

For many of the experiments you will also need to carry out some simple procedure on Wednesday and/or Friday - most of these take 10 minutes or less. You will be working in groups of 2-3; you and your partners can share the responsibility for carrying procedures during non-scheduled times. Pick partners with whom you can work well and with whom you can coordinate schedules.

Goals and expectations for the class as well as the syllabus will be reviewed. A brief overview of laboratory project will be given emphasizing the sequential nature of the experiments. Laboratory groups will be established.

Lecture: Introductory lecture explaining the use of Drosophila melanogaster and the enhancer trap methods.

Techniques: Basic laboratory techniques including laboratory safety, liquid handling, serial dilution and bacteria culturing techniques will be reviewed.
Behavioral testing to isolate mutants. Each group will receive a set of flies to flight test. Each group should find one flightless mutant line that is common to all groups. We will call this line the common mutant. Each set of flies will also contain several different lines of wild-type flies plus one other P-element insertion lines known to exhibit a mutant phenotype for which no gene has been identified.

Lecture: Review of reading and tutorial assignments. Discussion of reporter gene use. Introduction to the use of the laboratory computers. Introduction to Flybase.

Techniques: Start the procedure for determining the reporter gene expression pattern in adults and embryos of all mutants.

Lecture: DNA isolation and purification, restriction enzymes and their use, basics of cloning.

Techniques: Prepare chromosomal DNA from mutant lines and wild-type flies for use in the Plasmid Rescue and in Genomic Southern Analysis.

Complete the lacZ staining protocol. Analyze the lacZ staining patterns.

Lecture: Agarose gel electrophoresis, transformation techniques, discussion of plasmid vectors, phage vectors, cosmids and P1 clones, selection methods (antibiotic, blue/white, toxic proteins…)

Techniques: Precipitate and dry chromosomal DNA and then size fractionate by agarose gel electrophoresis. Digest chromosomal DNA from the mutant lines with appropriate restriction endonucleases. Make competent cells for next week’s laboratory. Introduction to the use of high speed and ultracentrifuges will be included in this laboratory.

Lecture: Plasmid DNA preparation (small scale and large scale, CsCl gradients and commercially available kits).

Technique: Pellet the ligated DNA by centrifugation. Transform competent XL1-Blue cells with ligated DNA.

Technique: Prepare plasmid DNA by alkaline lysis method. Size fractionate plasmid preparations by gel electrophoresis and set up double digests of plasmid preparations.

Lecture: Manual and automated sequencing techniques.

Technique: Size fractionate the plasmid digests by gel electrophoresis. This will separate vector from the attached genomic DNA. The genomic DNA is called the plasmid rescue fragment. The next logical step to carry out is sequencing of the plasmid rescue fragment. Since the entire Drosophila genome has been sequenced, sequencing of the fragment would tell you the location of the P-element insertion and whether or not the insertion is within a known gene. Automated sequencing data for the common gene is already available and will be provided. Use Flybase, the Berkeley Drosophila Genome Project (BDGP) database and the National Center for Biotechnology Information (NCBI) database to carry out blast searches of databases to determine whether the sequenced genomic fragments have homology to any known Drosophila sequences and/or to sequences in other species.

Sequence data is not available for plasmid rescue fragments for unknowns. You will analyze the gel of the digested plasmid DNA to determine which of the clones from the unknown mutants are likely to be "true" clones. These will be sent out for sequencing. Sequencing requires the use of highly purified DNA. Any contamination with phenol and chloroform will interfere with the sequencing reaction and obscure the reading of base pairs. Therefore, once positive clones have been identified, you will prepare plasmid DNA using a commercially available plasmid DNA isolation and column purification kit. To begin this process, start overnight cultures of the clones you want to sequence.

Begin the Total RNA Preparation by homogenizing adult flies from mutant lines and from wild-type lines in RNA homogenization buffer. Preparations will be left at 0˚C.

Lecture: RNA Preparation.

Technique: Prepare column purified plasmid rescue DNA from your unknown mutant. Run a test gel of your preparations. Also use UV spectrophotometry to analyze your DNA. Use of the spectrophotometer will be demonstrated. The DNA will be sent out for automated sequencing. When the sequencing data are available, you will carry out blast searches of databases to determine whether the sequenced genomic fragments have homology to any known Drosophila gene sequences and/or to sequences in other species. The database search using the sequence data from the genomic clones of the unknown mutants may lead to any of the following results:

Finish Total RNA preparation.

Digest chromosomal DNA (which was prepared during Laboratory 3) for Genomic Southern analysis (see Genomic Southern Analysis protocols).

Lecture: Genomic Southern and Northern analysis

Techniques: Size fractionate the digested chromosomal DNA by gel electrophoresis and blot onto nylon membrane. Poly A+ RNA preparations.

Lecture: PCR - Fundamentals and advanced techniques, PCR primers.

Techniques: Size fractionate RNA on a denaturing gel and blot onto nylon membrane. Use the Jellyfish and MacVector Programs to pick primers for PCR of a segment of the myosin heavy chain gene which includes exon 18.

Ralph Rapley and John M. Walker (eds) (1998). Molecular Biomethods Handbook. Humana Press, New Jersey.

Marjorie A. Hoy (1994).Insect Molecular Genetics: An Introduction to principles and Applications.Academic Press, San Diego.

Twyman,Richard M.(1997 ). Advanced Molecular Biology: A Concise Reference. Bios Scientific Publishers, New York.

Sambrook, Joseph and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Press, Cold Spring Harbor, New York.

You must keep a laboratory notebook and you must keep it up-to-date. Your laboratory notebook must have detachable carbon copies. Please do not keep your notes from lecture in your laboratory notebook; keep them in a separate notebook. Notebook entries for each laboratory session should include: 1) the date, 2) a heading to indicate which experiment the notes pertain to (Example, Northern Analysis, Labeling the DNA Probe), 3) a brief statement about the purpose and design of the procedure, 4) the steps in the procedure, including any problems you may have encountered and what you did about them, 4) results including drawing, tables and graphs, 5) conclusions including interpretations of the data, possible reasons for results other than those expected and things you might do to improve the experimental design.