# The toolbox

> The methods behind my projects, each explained in a sentence or two. Science is mostly careful measurement plus a good question.

Source: https://khushisingh.in/research/toolbox/
Author: Khushi Singh (khushisingh.in). Licence: CC BY 4.0 for text and original illustrations, unless a credit says otherwise.
Evidence labels used on this site: established, strong evidence, emerging, hypothesis, debated. Where a page has Simple, Curious and Expert versions, all three follow.

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Research · Methods

## Seeing

**Confocal microscopy**

A laser microscope that takes sharp optical “slices” through cells and tissues, so you can see one layer at a time and build 3D views.

**Single-molecule FISH (smFISH)**

Glowing DNA probes stick to one RNA type, so every single RNA molecule appears as its own bright dot.

**Immunostaining**

Antibodies, nature’s precise “grabbers”, are tagged with dyes to show where a protein or chemical mark sits.

**Live imaging of embryos**

Watching development happen in real time, gently, without fixing the sample.

**Genetically encoded sensors**

Proteins built into an animal that glow more or less depending on a molecule, so chemistry becomes visible.

## Changing genes

**GAL4/UAS**

A two-part genetic switch from yeast. Turn a gene on only in the tissue you choose.

**RNA interference (RNAi)**

Turn a gene down by destroying its messenger RNAs.

**Mutants and genetic crosses**

Classic fly genetics: combine traits through careful breeding, tracked with visible markers such as curly wings.

**Cloning and site-directed mutagenesis**

Cut, paste and edit DNA to make a protein with exactly the change you want.

## Working with proteins

**Protein purification**

Make a protein in bacteria, then fish it out using a molecular “handle” (affinity) and charge (ion exchange).

**Fluorescent labelling**

Attach a dye at one precise spot, so the protein reports what it is doing.

**Spectrophotometry**

Measure how a sample absorbs or emits light to count molecules or watch droplets form.

**Enzyme kinetics**

Measure how fast an enzyme works at different fuel levels to learn how it behaves (Michaelis–Menten).

**Western blotting**

Separate proteins by size and detect specific ones with antibodies.

## Measuring behaviour

**Drosophila Activity Monitor (DAM)**

Each fly lives in a small tube and crosses an infrared beam as it walks; counts per minute give activity, sleep (five or more minutes still) and daily rhythms.

**Fertility assays**

Count eggs laid and offspring that hatch, to see whether a change matters for the animal.

## Making sense of data

**Image analysis (Fiji/ImageJ)**

Turn pictures into numbers: count, measure intensities, and compare fairly with the same settings.

**Python and R**

Reproducible analysis pipelines and statistics, from raw fluorescence to fitted curves.

**Good practice**

Calibrations, blanks, replicates and honest error bars, plus knowing what a result does not show.