Lab 03B HHMI Bacterial Identification Virtual Lab 2026 – 85 Exam Questions & Answers – 16S rRNA, PCR, Gel Electrophoresis & BLAST

This document contains approximately 85 structured exam questions and expert-verified answers for Lab 03B HHMI Bacterial Identification Virtual Lab 2026. It provides comprehensive coverage of bacterial identification using the 16S rRNA gene, explaining how identification relies on sequencing the small subunit ribosomal RNA (16S rDNA) and matching it against established genetic databases. The guide thoroughly outlines bacterial DNA extraction procedures, including digestive buffer treatment, heat denaturation at 100°C, centrifugation, and transfer of the DNA-containing supernatant for PCR amplification. The material offers an in-depth explanation of polymerase chain reaction (PCR), including Master Mix components (buffer, nucleotides, primers, heat-stable DNA polymerase), positive and negative controls, and thermocycler parameters (initial denaturation, 30 melt-anneal-extend cycles, final extension, 4°C hold). It details the purpose of each PCR step (95°C denaturation, 60°C annealing, 72°C extension), microconcentrator column purification, and recovery of amplified 1500 bp 16S rDNA fragments. Additionally, the document explains DNA sequencing preparation using fluorescence-tagged dideoxynucleotides, primer-specific amplification, and capillary gel electrophoresis in an automatic sequencer. It clarifies the principles of gel electrophoresis, fluorescent detection, variable-length fragment analysis, conserved versus variable regions, E-values, and the use of BLAST (Basic Local Alignment Search Tool) for sequence comparison. The lab also includes identification of unknown bacterial samples such as Bartonella henselae, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Yersinia pestis, and Yersinia enterocolitica, along with their clinical specimen sources. This resource is particularly suitable for: Microbiology students Molecular Biology students Biotechnology students Pre-med and pre-health majors Laboratory science students AP Biology and advanced biology students It is relevant for courses such as: Microbiology Lab Molecular Genetics Laboratory Biotechnology Techniques Bacterial Identification and Diagnostics Cell and Molecular Biology Clinical Microbiology Keywords: HHMI Bacterial Identification Lab 2026, 16S rRNA Sequencing, Polymerase Chain Reaction PCR, PCR Thermocycler Steps, DNA Extraction Procedure, Microconcentrator Column Purification, Dideoxynucleotides, DNA Sequencing, Gel Electrophoresis, Capillary Sequencer, BLAST Analysis, E Value Interpretation, Conserved Regions, Variable Regions, Agrobacterium Not Included, Bartonella henselae Identification, Escherichia coli Sample, Pseudomonas aeruginosa Urine Sample, Salmonella typhimurium Blood Sample, Yersinia pestis Sputum Sample