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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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