Molecular Techniques: A Project-Based Undergraduate Laboratory

Author(s): Ming-Mei Chang

ISBN: 978-1-956862-23-2

Affiliation: SUNY Geneseo

🔒Instructor Resource: Appendix C: Instructor Key for Solutions and Dilutions (password required, email ost@geneseo.edu for access): 

About the Book

Molecular biology, examining the structures and functions of nucleic acids and proteins, is crucial to advancing biological science and improving human life, particularly in agriculture and medicine. Advances in the field can transform a crop to resist disease or uncover clues to fight illness by manipulating DNA. Techniques for molecular biology research and applications have advanced rapidly over the past decades, and undergraduates need hands-on experiences in this vital field of science. This text provides a structured approach to learning and practicing those techniques, covering DNA, RNA, and protein across five modules and is tailored for a one-semester undergraduate lab course. Each module extends 2–5 weeks of lab exercises and features:

1. An aim and overview introducing the project’s topic, principles, and concepts.
2. A flowchart summarizing weekly lab exercises with estimated time frames.
3. A list of required equipment and materials to help instructors prepare.

Each lab exercise begins with an objective(s), an introduction to the techniques used, and detailed procedures for the corresponding experiment; data analysis and discussion at the end enhance students’ comprehension of the content. Students will become familiar with techniques widely used in molecular biology research, such as degenerate PCR cloning, Southern blot, probe labeling, bacterial recombineering with and without CRISPR-Cas9, RT-qPCR, SDS-PAGE, and Western blot. Most lab exercises were adopted from published research projects to ensure the feasibility and success of the experiments conducted by inexperienced undergraduates. All modules have been taught to biochemistry and biology 3^rd– and 4^th-year students at SUNY Geneseo in multiple sections of the Molecular Techniques Lab over several years. Most lab exercises, along with student results, were published previously in Biochemistry and Molecular Biology Education.

Table of Contents

• Introduction
• To the Students
• To the Instructors
• Laboratory Safety
• Pipetting
• Making Solutions and Dilutions
• Weekly Laboratory Notes
• Laboratory Report
• Module 1: Degenerate PCR and Topoisomerase (TOPO)-based DNA Cloning
  • Overview
  • Equipment and Materials
  • Week 1: Genomic DNA Isolation and Quantification; Degenerate PCR
  • Week 2: Agarose Gel Electrophoresis; Purification and Quantification of PCR Products; Degenerate PCR Primer Design
  • Week 3: TOPO-based DNA Cloning of Nucleotide Binding Site (NBS) Sequences; Bacterial Transformation
  • References
• Module 2: Analyzing Cloned NBS Sequences
  • Overview
  • Equipment and Materials
  • Week 1: Plasmid DNA Isolation and Quantification; Restriction Digestion; Sample Preparation for DNA Sequencing
  • Week 2: Agarose Gel Electrophoresis; Restriction Mapping; Southern Transfer
  • Week 3: DNA Extraction from Agarose Gel and Quantification; PCR Digoxigenin Probe Synthesis
  • Week 4: Southern Blot Hybridization and Detection
  • Week 5: Web-based Sequence Analysis of Cloned NBS Sequences
  • References
• Module 3: Gene Editing by Recombineering With or Without CRISPR-Cas9
  • Overview
  • Equipment and Materials
  • Week 1: Recombineering of Escherichia coli
  • Week 2: Validation of Recombineering-edited Escherichia coli by Colony PCR and Web-based Sequence Analysis
  • Week 3: CRISPR-Cas9-assisted gene editing in Escherichia coli
  • Week 4: Validation of CRISPR-Cas9-assisted Gene-editing in Escherichia coli by Colony PCR and Web-based Sequence Analysis
  • References
• Module 4: Studying Gene Expressions by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
  • Overview
  • Equipment and Materials
  • Week 1: Total RNA Isolation, Quantification, and Integrity Assessment
  • Week 2: RT-qPCR
  • References
• Module 5: Exploring the Sensitivity and Specificity of Western Blot
  • Overview
  • Equipment and Materials
  • Week 1: Protein Isolation and Quantification for SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE)
  • Week 2: SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Transfer
  • Week 3: Immunodetection of Western Blot for Bovine Serum Albumin (BSA) Protein
  • References
• Appendix A: Working Solutions for Immunodetection of Southern blot
• Appendix B: General Remarks on Handling RNA
• Appendix C: Instructor Key for Solutions and Dilutions 🔒

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Biology, Forthcoming, Natural Sciences