Unit 19: Experimental Methods in Cell Biology
The tools that let anyone see, sort, copy, cut, or read what a cell is made of and doing — from a microscope's resolution limit to editing a genome on purpose.
9 topics · 36 questions
Each topic is a path, not a menu: work down it in order and every question builds on the one above. Click any of them to watch the answer draw itself.
Light and Fluorescence Microscopy
- How it worksHow does the wavelength of visible light set a hard physical limit on a light microscope's resolution?
- How it worksHow does fluorescence microscopy use a specific dye and filtered light to make one structure glow against a dark background?
- Common mistakeWhy isn't magnification the same thing as resolution when judging how good a microscope image is?
- Worked exampleFind the resolution limit of a light microscope using 450 nanometer blue light with a numerical aperture of 1.4.
Your turn1 of 2
Find the resolution limit of a light microscope using 609 nanometer light with a numerical aperture of 1.119.Electron Microscopy: TEM and SEM
- How it worksHow does using a beam of electrons instead of light push resolution down to the scale of individual proteins?
- How it worksHow does a transmission electron microscope image a thin slice, while a scanning electron microscope instead images a surface by bouncing electrons off it?
- Common mistakeWhy can't electron microscopy be used to watch a living cell moving in real time?
- Going furtherWhat happens to the detail visible in a cryo-electron micrograph when the sample is flash-frozen instead of chemically fixed?
Your turn1 of 2
Electron microscopy reveals extraordinary detail in a fixed cell sample, down to the scale of individual proteins. A student wants to use this same technique to directly observe a living cell moving and dividing in real time. Why can't electron microscopy be used to watch a living cell moving in real time?Cell Fractionation by Differential Centrifugation
- How it worksHow does differential centrifugation separate organelles by spinning a homogenate at increasing speeds?
- How it worksWalk me through why the nucleus pellets out first, mitochondria next, and ribosomes only at the very highest speeds.
- Common mistakeWhy isn't cell fractionation just crushing a cell and hoping its parts happen to end up in separate tubes?
- Worked exampleFind which organelle pellets first between one sedimenting at 1,000 times gravity and one requiring 100,000 times gravity.
Your turn1 of 2
A homogenized cell sample is spun in a centrifuge, first at a relatively low speed and then, using the leftover supernatant, at a much higher speed. Between an organelle that pellets out at 1,000 times gravity and one that requires 100,000 times gravity to pellet, which one is larger and denser?Gel Electrophoresis of Proteins and Nucleic Acids
- How it worksHow do smaller DNA fragments migrate faster through a gel's mesh of pores than larger fragments under an electric field?
- How it worksHow does SDS coat every protein in a uniform negative charge so a protein gel separates purely by size?
- Worked exampleFind which DNA fragment migrates farther on a gel after the same run time: one 500 base pairs long or one 5,000 base pairs long.
- Common mistakeWhy isn't gel electrophoresis separating molecules by their natural charge alone, without any help from the technique?
- Going furtherWhat happens to band resolution on a gel when the agarose concentration is increased?
Your turn1 of 2
Two DNA fragments, one 500 base pairs long and one 5,000 base pairs long, are loaded into neighboring wells of the same agarose gel and run for the same amount of time under the same electric field. Which fragment migrates farther through the gel?The Polymerase Chain Reaction
- How it worksHow do primers, a heat-stable polymerase, and repeated heating and cooling cycles double a DNA sample with each round?
- Worked exampleFind how many copies of a DNA sequence exist after 10 cycles of PCR starting from a single template molecule.
- Common mistakeWhy isn't PCR sequencing the DNA it amplifies, since both methods work with the same molecule?
- Going furtherWhat happens to PCR amplification when the primers are designed to bind two sites 10 kilobases apart instead of 500 bases apart?
Your turn1 of 2
Find how many copies of a DNA sequence exist after 22 cycles of PCR starting from a single template molecule.DNA Sequencing
- How it worksHow does Sanger sequencing use chain-terminating nucleotides to read out a DNA sequence one base at a time?
- How it worksHow does next-generation sequencing read millions of short DNA fragments in parallel instead of one long read at a time?
- Common mistakeWhy isn't DNA sequencing the same technique as PCR, even though both start from the same kind of sample?
- Worked exampleFind the minimum number of 150-base sequencing reads needed to cover a 3 billion base pair genome at 30-fold coverage.
Your turn1 of 2
Find the minimum number of 239-base sequencing reads needed to cover a 2771479000 base pair genome at 47-fold coverage.Immunostaining and Antibody-Based Detection
- How it worksHow does a primary antibody bind its target protein while a fluorescently labeled secondary antibody makes that binding visible?
- How it worksWalk me through how a Western blot combines gel electrophoresis with antibody detection to identify one protein in a mixture.
- Common mistakeWhy isn't an antibody's specificity guaranteed to be perfect, with zero chance of binding the wrong protein?
Your turn1 of 2
An antibody is chosen to specifically detect one particular protein among the tens of thousands present in a cell sample, and it is generally assumed to bind only that intended target. A student assumes this specificity must be absolutely perfect, with zero chance of the antibody ever binding an unintended protein. Why isn't an antibody's specificity guaranteed to be perfect, with zero chance of binding the wrong protein?Green Fluorescent Protein as a Reporter
- How it worksHow does GFP's own amino acid chain fold into a barrel that generates green light without needing any other cofactor?
- How it worksHow does fusing the GFP gene to a gene of interest let researchers watch that protein move inside a living cell in real time?
- Common mistakeWhy isn't a GFP fusion protein just a dead label sitting next to the protein of interest, since it supposedly doesn't affect the protein's behavior?
- Going furtherWhat happens to an experiment's usefulness when a GFP tag is fused at a site that disrupts the studied protein's folding?
Your turn1 of 2
Researchers routinely fuse the GFP gene to a gene of interest and then track the resulting protein moving inside a living cell, treating GFP as if it has no real effect on the protein it's attached to. A student assumes GFP must simply be a completely inert, dead label sitting harmlessly next to the protein of interest. Why isn't a GFP fusion protein just a dead label sitting next to the protein of interest, since it supposedly doesn't affect the protein's behavior?CRISPR-Cas9 Genome Editing and Model Organisms
- How it worksHow does a guide RNA direct the Cas9 enzyme to cut DNA at one exact matching sequence?
- How it worksHow does a cell's own double-strand break repair machinery get hijacked by CRISPR to insert or disrupt a gene?
- Common mistakeWhy isn't CRISPR guaranteed to only cut its one intended target sequence and nowhere else in the genome?
- Ties togetherHow does choosing a model organism like a mouse, a fly, or yeast connect to which CRISPR-edited questions about human genes can actually be tested?
Your turn1 of 2
A guide RNA is designed to direct Cas9 to cut one specific, intended sequence in a genome. A researcher assumes this guarantees Cas9 will cut only that exact intended sequence and nowhere else. Why isn't CRISPR guaranteed to only cut its one intended target sequence and nowhere else in the genome?