MMG104: Introduction to Recombinant DNA Technology

Coordinator: Stephanie Phelps

Offered each Spring Section A: M & W 9:35 to 12:35

Section B: M & W 12:50 to 3:50

Introduction to the basic principles and techniques used in Recombinant DNA technology.

Techniques performed include:

Restriction digests	Yeast tetrad analysis and the halo assay
Electrophoresis	Yeast complementation and mating
Restriction mapping	Yeast 2-hybrid
Ligations	Mammalian cell subculturing
Bacterial Transformations	Eukaryotic transfections using GFP
Colony PCR	Fluorescent microscopy
Plasmid Isolation	PCR-RFLP analysis at 2 loci
Read a sequencing chromatograph	DNA profiling using 4 CODIS STRs
BLAST searches	Alu Element Insertion analysis at 2 loci

2009 Schedule

2009 Grading

MMG201: Molecular Cloning Lab

Coordinator: Stephanie Phelps

Offered each Fall Section A: T & Th 1 to 5:15

Section B: M & W 11:45 to 3:50

An intensive one semester advanced laboratory course in which students will learn the fundamentals of recombinant DNA technology through the isolation and characterization of a eukaryotic gene. 

DNA/Cloning:

The plasmid containing a truncated p53 cDNA must be modified to clone in a full-length p53 driven by the GAL 1/10 promoter.

RNA:

To understand how p53 acts as a transcription factor and how it can have varying affinities for different enhancer sequences, the p53 plasmid and the enhancer/reporter plasmids will be transformed into yeast. Total RNA will be isolated and analyzed for the amount of lacZ (reporter) mRNA made.

Protein:

We will examine the DNA binding domain (DBD) of p53 and how mutations introduced to that region can influence its ability to act as a transcription factor. We will be mutagenizing the DBD via PCR and then recloning the full-length cDNA by homologous recombination in yeast. Mutant and wild-type p53’s will be analyzed by Western blotting.

Techniques performed include:

Restriction Digests	In-plate Β-Galactosidase Assay
Use of Purification Columns	Isolation of RNA from Yeast
Isolation of DNA from Gel	First Strand cDNA Synthesis
Ligations	Quantitative RT-PCR
Yeast Transformations	Liquid Β-Galactosidase Assay
Β-Galactosidase Assay on Media & Filter Lifts	Mutagenic PCR
Plasmid Isolation from Yeast	Isolation of Protein from Yeast
E. coli Transformations	Cycle Sequencing
Plasmid DNA Isolation	Sequence & Mutation Analysis
Making Yeast Competent Cells	Immunoprecipitation
Nucleic Acid Quantitation	SDS-PAGE Gels/Westerns
PCR	ECL Immunodetection

2008 Schedule

2008 Grading

MMG 203: Mammalian Cell Culture in Molecular Biology

Coordinator: Stephanie Phelps

Offered Every Other Spring

2009 – T & Th 11:30 to 3:45

Students will learn the basic principles and techniques of mammalian cell culture as well as cell and mammalian molecular genetics. 

Techniques performed include:

Setting up a cell culture lab	Transfection with pEGFP-F
Medium making	FLOW analysis of the cell cycle
Cell subculturing	siRNA Transfection
Enumeration	Myoblast differentiation
Cell viability using PI and FLOW cytometry	Transfection with myoD
Growth curves	Indirect Immunofluorescence
Metaphase spreads & karyology	Cell fate switching: Making muscle into bone and making fat from fibroblasts
Mycoplasma detection	RT-PCR
Cryopreservation and thawing cells	Primary cell culture of Hair Follicles

2009 Schedule

2009 Grading

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Stephanie.Phelps@uvm.edu

Revised: February 2009