> [!question] > How would a DNA strand with epigenetic markers (ex: methylation) appear on a gel? # Gel Electrophoresis Electrophoresis is the movement of charged molecules toward an electrode of an opposite charge. - In gel electrophoresis, a gel matrix (such as agar or polyacrylamide) is used as the matrix. ## Analyzing DNA/RNA At alkaline pH (7-14), DNA and RNA are *negatively-charged* and will migrate in an electric field. Unlike [[SDS-PAGE]] (protein electrophoresis), the *phosphate groups* present within DNA/RNA provide a natural *charge-to-mass ratio*, meaning that . **Acrylamide gels** are used for smaller fragments (<200 bp) - Useful for sequencing ### Agarose Gels **Agarose gels** are the most common matrix used when running fragments larger than 200 bp through an electrophoresis. - A polysaccharide (galactose/galactopyranose) found in red seaweed which readily polymerizes into a thick, porous gel #### Changing Gel Resolution #### Changing Gel Resolution | % Agarose | Resolution | | --- | --- | | 0.5% | 1000 bp - 30 kb | | 0.7% | 800 bp - 12 kb | | 1.0% | 500 bp - 10 kb | | 1.2% | 400 bp - 7 kb | | 1.5% | 200 bp - 3 kb | | 2.0% | 50 bp - 2 kb | Several *buffers* can be added to the gel mixture with varying benefits (mix and match to your pleasure): - **Tris-acetate-EDTA (TAE):** Often runs faster - **Tris-borate-EDTA (TBE):** Often better resolution Because agarose gels can be *significantly thicker* than acrylamide gels they do not need a separate *stacking gel* and can also be run **horizontally**. The **sample loading buffer** often contains multiple tracking dyes since the rate at which they travel through the gel can vary depending on the concentration of the gel used. - *Formaldehyde* is often required when running RNA to keep it denatured. A [[Northern Blot]] can often follow --- > [!important] Impact of [[Nucleic Acid Structure|DNA Conformations]] > The conformation of DNA within a gel can greatly affect the rate at which it travels through a gel (often based on its surface area). From fastest to slowest: > 1. Circular ssDNA > 2. *Supercoiled* (depends on the degree of supercoiling) > 3. *Linear* > 4. *Relaxed Circular* > 5. *Nicked Circular* (plasmids with a single ssDNA cut and the other strand intact) > 6. *Catenated (Linked) Plasmids* > > This is often avoided by digesting all DNA to ensure that it is linear for comparisons ### Pulse-Field Gel Electrophoresis > zig zag shit While standard electrophoresis is limited to ~50 kb, much larger DNA fragments (up to 1 mbp) can be separated in an agarose gel by using multiple electrodes to vary the current direction in **pulse-field gel electrophoresis.** ![[Gel Electrophoresis.png|300]] ### Visualization Techniques One of the simplest techniques to visualize DNA/RNA samples on a gel is through **UV shadowing**. Because [[nucleic acids]] absorb UV light (at 260 nm), passing UV light through a gel will result in shadows appearing on surfaces where the gel contains nucleic acid bands. - Not nearly as practical as modern techniques/far too basic ![[UV Shadowing of DNA and RNA.png|400]] --- **Ethidium Bromide (EtBr)** is a fluorescent dye that will intercalate/insert itself within nucleic acids and is much more common for DNA and dsRNA visualization. - Can be added to the gel or soaked in afterwards - Considered a carcinogen/hazardous ![[Ethidium Bromide Usage Example.png|400]] --- An alternative to ethidium bromide staining is the use of special dyes like **SYBR Green** or **SYBR Gold**, which also intercalate DNA and interact with the minor groove and phosphate backbone. ![[Gel Electrophoresis-2.png|400]] (it just looks pretty) --- Unlike the previous techniques, **Methylene Blue** and **Crystal Violet** can be visualized in *real-time* as the sample is travelling through the gel without needing to be developed first. - They are significantly less sensitive than the other options (only detecting $\micro$g of DNA) ![[Gel Electrophoresis-3.png|400]] --- **Autoradiography** is the most sensitive technique for visualizing ## Analyzing Proteins > See: [[SDS-PAGE]] ### Isoelectric Focusing By using a pH gradient you can have specific proteins stop moving when they reach the pH associated with their pI (neutral state) as they will no longer be pulled to either the negative or positive charge