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: plasmids and others
• Gene selection techniques and cloning strategies
• Genomic library construction
• cDNA library construction
• Restriction enzymes and ligase enzymes for cloning DNA
• DNA and RNA manipulations in vitro
• Polymerase chain reaction (PCR)
• Reverse transcriptase PCR (RT-PCR)
• Real-time PCR and quantitative RT-PCR
• Gel electrophoresis (agarose and PAGE)
• DNA, RNA and protein isolation and purification
• Western blot analysis
• Gene expression in prokaryotes and eukaryotes
• Methods for studying gene expression
• DNA and RNA hybridization
• Sanger (dideoxy) DNA sequencing
• Thermal cycle sequencing
• Next-Generation Sequencing (NGS)
• Computer analysis of DNA, RNA and protein sequences
• Expression vectors in E. coli
• Gene expression/protein production in heterologous hosts
• Genome projects
• RNAseq
• DNA fingerprinting and microsatellites
• RNA interference
• MicroRNA and other small regulatory RNAs
• Human genetic analysis
• DNA methylation and gene expression
• CRISPR/Cas9 and gene editing

Summary of laboratory experiments

This intensive two-week course emphasizes hands-on molecular biology laboratory work. Participants will average approximately five hours each day working at the bench and five hours in lecture. All the research is hands-on; there are no demonstrations. All of the techniques are woven into six cohesive research projects carried out by each participant during the two-week course. Lectures/discussions focus on the application of these methods in molecular biology research.

Experiment #1: Gene Cloning and Protein Expression

• 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 cloned 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

• Prepare a Next-Gen cDNA library
• Next-Gen Illumina MiSeq DNA Sequencing
  
  

Experiment #5: CRISPR/cas9 Gene Editing

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

Experiment #6: Human DNA/Genetic Analysis

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

Weekly schedule