Gene Cloning: PCR and Restriction Cloning
Description
In this virtual lab, students clone a green fluorescent protein gene (demoGFP) into a plasmid vector, working through the full workflow of a real molecular biology lab. They design primers with restriction-site tails and assemble a PCR reaction by pipetting each reagent. Next, they program a thermocycler and watch amplification in real time, then cast, load, run, and image an agarose gel to check the product. In the second part, they search a library of 35 restriction enzymes to find suitable cutting sites, digest the PCR product and vector, and ligate the insert using appropriate molar ratios. Finally, they transform competent E. coli by heat shock and identify recombinant colonies through blue-white screening. Every choice has realistic consequences, such as faint bands, failed digests, or overcrowded plates, and "Why?" explanations help students understand what went wrong and how to fix it. The lab can be completed in Guided or Challenge mode and ends with a 12-question assessment.
Learning Outcomes
By the end of this lab, students will be able to design PCR primers with appropriate melting temperatures, GC content, and added restriction sites. They will be able to assemble a PCR reaction with the correct volumes and concentrations and explain the purpose of each component. They will also set up and justify a thermocycler program covering denaturation, annealing, and extension. Students will be able to prepare and run an agarose gel, estimate DNA fragment sizes against a ladder, and troubleshoot common problems such as faint or missing bands. They will select restriction enzymes by identifying recognition sites in a DNA sequence and predict the resulting fragment sizes and end types. Students will also explain how vector-to-insert molar ratios and dephosphorylation affect ligation success. Finally, they will carry out heat-shock transformation and interpret blue-white screening to identify recombinant clones, connecting each step of the workflow to the molecular principles that make gene cloning possible.