To the Instructors

This lab book, Molecular Techniques, is designed to provide a selection of alternative lab exercises that supplement or comprise a one-semester molecular techniques lab course, targeting 3^rd– and 4^th-year biochemistry, biology, and related science majors. All labs described in this book had been taught for several years in multiple sections of a two-credit, four-hour Molecular Techniques Laboratory (Biol 390) at SUNY Geneseo. Each lab section accommodates 12–18 students working in pairs. The majority of students taking the lab were 4^th-year Biochemistry students.

To refresh students’ basic laboratory skills and ensure a smooth start to the semester, it is recommended that students review “Laboratory Safety,” “Pipetting,” “Making Solutions and Dilutions,” “Weekly Laboratory Notes,” and “Laboratory Reports” during the first week of the semester. In addition to specific instructors’ notes in individual lab exercises, the following notes and suggestions are provided for preparing and teaching the labs.

Organism establishment and maintenance

The organisms used are grapevine (Vitis spp.) (Modules 1 and 2), E. coli (Modules 1, 2, and 3), A. thaliana (Module 4), and fruit fly (Drosophila melanogaster) (Module 5), all of which are easy to grow and maintain. E. coli, A. thaliana, and fruit flies are model organisms for teaching and research. Instructions for their establishment and maintenance were previously described in published papers, available for instructors’ reference.^1-5 Briefly, grapevines can be grown in the greenhouse or field; young leaves are harvested, then immediately frozen in liquid N₂ and stored at -70°C from late spring to early summer.¹ Various E. coli strains must be created or purchased, and then cultured prior to the corresponding experiments.³ Glycerol stocks of E. coli strains should also be made and stored at -70°C.^1,3 A. thaliana is grown in a growth chamber, treated, and harvested, followed by instant freezing in liquid N₂ and storing at -70°C.⁴  Populations of fruit flies need to be established days before the lab starts and expanded for multiple lab sections. Detailed procedures for the establishment and maintenance of the fruit fly were described in the previously published paper.⁵

Timing

Although most labs can be completed within a four-hour lab session, some require students to work outside the lab. For example, it takes time for bacteria to form colonies and cultures to reach specific cell densities to complete the experiments. Modules 2 and 3 each include steps that must be completed outside of lab time. For these assignments, be sure to indicate the dates of these labs in the syllabus and remind students at the beginning of the semester and the week prior to the corresponding lab.

Module 2 Week 1 includes setting up an overnight bacterial culture for plasmid isolation and stopping restriction digestion. Week 2 requires disassembling the Southern transfer, and Week 3 requires stopping and storing the PCR-amplified Dig probe, each taking approximately 10 minutes. In the evening prior to the Week 4 lab, students need to set up a 30-minute Southern prehybridization and an overnight hybridization. These steps should be conducted synchronously under the instructor’s supervision in the lab.

In Module 3 Weeks 1 and 3 the recombineering and CRISPR/Cas9-assisted recombineering labs require that bacterial cultures be incubated for ≥ 3 hours after electrotransformation. Students must wait before diluting and plating the bacteria, and then incubate the plates overnight. The plates should be sealed and stored in the refrigerator the next day. Bacterial dilution and plating take approximately 30 minutes, and sealing and refrigerating the plates take approximately 10 minutes.

Flexibility

The lab modules can be modified and rearranged based on material availability and the course schedule. For Module 1, students can clone NBS sequences from other plant species without redesigning the primers, because the primers correspond to highly conserved amino acid sequences in the NBS region originally designed to clone NBS from different plant species. For Southern blot analysis in Module 2, students can use their cloned DNA inserts for Dig probe synthesis instead of the known NBS DNA, as the identities of the cloned DNAs can be validated through outsourcing DNA sequencing and web-based sequence analysis later. Module 2 can also be shortened to two weeks by removing the Southern blot analysis sections from Weeks 2 and 3 and combining the remaining Week 2 procedures (excluding Southern transfer) with Week 4’s web-based sequence analysis. See an example of this configuration in Table 1. In this case, Module 3 can be added.

Also Module 3 recombineering can be taught without CRISPR-Cas9 if time is constrained. However, adding CRISPR-Cas9 to recombineering makes the project challenging enough to be an upper-level lab. Alternatively, students can conduct CRISPR/Cas9 gene editing using commercially available kits, such as Bio-Rad’s CRISPR/Cas9 gene editing lab, which disrupts the lacZ gene and produces white colonies upon successful editing.⁶ In that case, students learn two different gene editing approaches separately.

In addition to studying the effect of dark treatment on the expression of two RubisCO small-subunit genes in Module 4, students can investigate the impact of dark treatment duration on the expression of other photosynthetic genes. Since the A. thaliana genome is well-characterized, corresponding qPCR primers for RT-qPCR can be designed easily. For Module 5, if the purpose is to demonstrate the specificity and sensitivity of Western blot analysis rather than to investigate the presence of BSA and its analogs in fruit fly hemolymph, commercially available unrelated, non-BSA proteins can be used without protein isolation steps.

Each module and weekly lab exercise provide project aims and objectives, respectively, enabling instructors to select modules that align with their curricula. The order of the modules can be rearranged, but the order of weekly lab exercises within each module must remain the same. For example, Lab Schedule A Modified (Table 2) differs from Lab Schedule A (Table 1) by swapping Modules 3 and 4, with Module 4 conducted before the more complex Module 3, which is scheduled for the end of the semester, when students feel more comfortable working in the lab.

Since the book includes more weekly labs than there are weeks in a semester, it allows instructors to rotate through certain modules across different semesters. For example Lab Schedule A (Table 1) and Lab Schedule A Modified (Table 2) removes the Southern blot analysis from Module 2 and replaces it with Module 3. Lab Schedule B (Table 3) keeps all lab exercises of Module 2 and contains Module 5 (Western Blot), without Module 3. Finally, if time permits, an additional week may be added at the end of each module to facilitate data analysis and discussion, address students’ questions, clarify any confusion, and correct any lab errors.

References