Molecular Biology Summer Workshop:
Lecture Topics and Laboratory Experiments

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Lecture topics

Below is a list of lecture topics. Some of these topics will be combined into a single lecture, while others will take several lectures.

• Cloning vectors: bacteriophages, plasmids and many others
• Genetic selection techniques and cloning strategies
• Genomic library construction
• cDNA library construction
• Restriction enzymes and ligase enzymes for cloning DNA
• Cloning in cosmids, YAC and BAC vectors
• Dna and rna manipulations in vitro
• Polymerase chain reaction (PCR)
• Reverse transcriptase pcr (RT-PCR)
• Quantitative PCR and quantitative RT-PCR
• Gel electrophoresis (agarose and page)
• DNA, RNA and protein isolation and purification
• Southern, northern and western blot analyses
• Gene expression in prokaryotes and eukaryotes
• Methods to study gene expression
• DNA and RNA hybridization
• Chain termination (dideoxy) DNA sequencing
• Thermal cycle sequencing
• Next-gen sequencing (NGS)
• Computer analysis of DNA, RNA and proteins/bioinformatics
• Expression vectors in E. coli
• Gene expression/protein production in heterologous hosts
• Genome projects
• Microarrays and RNA-seq
• Dna fingerprinting and microsatellites
• RNA interference (RNAi and siRNA)
• Microrna and other small regulatory RNAs
• Human genetic analysis
• CRISPR/Cas9

Laboratory experiments

This intensive two-week course emphasizes hands-on molecular biology laboratory work. Participants will spend approximately eight hours each day working at the bench. All the research is hands-on; there are no demonstrations. With 8 instructors and staff, the student to staff ratio is 6:1.

All techniques are woven into a cohesive research project carried out by each participant during the two-week course. Lectures and discussion sessions pertain to the application of these methods in molecular biology research.

Experiment #1: Gene Cloning and Protein Expression (Blue)

• cDNA cloning of the mouse GAPDH gene in a plasmid expression vector (pMAL)
• First and second strand cDNA synthesis and PCR to synthesize the GAPDH gene
• Ligation of the GAPDH cDNA into the plasmid expression vector pMAL
• Transformation of E. coli, selection of recombinant clones and DNA sequencing
• Expression and purification of the GAPDH fusion protein in E. coli
• Measure protein concentration and analyze on PAGE protein gels
• Western blot to specifically detect the GAPDH fusion protein

Experiment #2: Genome Analysis

• Isolate and purify genomic mouse DNA from liver tissue
• Amplify the transthyretin (Ttr) gene using the polymerase chain reaction (PCR)
• Analysis of the methylation state of Ttr and Rvt genes in mouse genomic DNA

Experiment #3: Gene Expression Analysis

• Preparation of total RNA from mouse liver tissue
• Amplification of Ttr mRNA by reverse transcriptase PCR (RT-PCR)
• RT-qPCR using real-time analysis (SYBR^® and TaqMan^® systems)

Experiment #4: Next-Generation Sequencing

• Using total RNA from mouse tissue (Expt. 3) prepare a next-gen cDNA library
• Sequence the library on the next-gen Illumina MiSeq DNA Sequencer
• Bioinformatics to analyze millions of DNA sequence reads

 
Experiment #5: CRISPR/cas9 Gene Editing in Yeast

• Design a CRISPR/cas9 gene editing plasmid for yeast and amplify in E. coli
• Isolation and purification of the pCAS/ADE2 sgRNA plasmid
• Construct a barcode/editing DNA fragment using PCR
• Co-transform yeast with the pCAS plasmid and the barcode/editing DNA fragment
• Demonstrate successful genome editing by phenotype and genotype

Experiment #6: RNA Interference in C. elegans

• Grow C. elegans then isolate and purify eggs to produce a synchronous culture
• RNA interference by feeding C. elegans on E. coli containing Bli DNA
• Observe the RNA interference Bli phenotype in adult C. elegans

Experiment #7: Human DNA/Genetic Analysis

• Isolation of your own DNA from cheek cells from your mouth
• PCR amplification of your own DNA for DNA fingerprint analysis
• PCR amplification of your taster gene: compare phenotype and genotype

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