# Goal/Experiment:
To prepare and use a 2% agarose gel for the separation and analysis of DNA fragments through gel electrophoresis.
# 2% Agarose Gel
**Author:** George Testo
**Affiliation:** The Pathogen & Microbiome Institute
**Date:** May 31, 2022
**Protocol Shared via:** [protocols.io link](https://protocols.io/view/2-agarose-gel-cac7szan)
**Date of Protocol:** Jun 03, 2022
**Disclaimer:**
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
## Introduction
Agarose gel electrophoresis is a method used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The molecules are separated by applying an electric field to move the charged molecules through an agarose matrix, with separation primarily based on size.
Routinely, agarose gels are suitable for separating DNA of various size ranges, can be stained and visualized under UV light, and DNA fragments can be extracted from the gel easily.
## Reagents
- 1 x DI Water
- 1 x bottle of TAE (or TBE)
- 1 x bottle of Agarose Powder
- 1 x bottle of SYBR Safe
- 1 x bottle of 6x Loading Dye
## Supplies
- Green temporary seal(s)
- Foil seal(s)
## Equipment
- 10µL pipette, tip box, & tips
- 20µL pipette, tip box, & tips
- 200uL pipette, tip box, & tips
- 1000uL pipette, tip box, & tips
- Serological pipette & 10mL tip
### Note on SYBR™ Safe Dye:
SYBR™ Safe DNA gel stain showed no or very low mutagenic activity in tests and is not classified as hazardous waste under U.S. Federal regulations. However, standard care should be taken when handling and disposing of this reagent in compliance with all local regulations.
#### Storage:
SYBR™ Safe DNA gel stain can be stored between 2°C to 25°C. SYBR™ Safe in DMSO freezes at low temperatures; it must be completely thawed and mixed before use.
## Procedure
### Pouring a Standard 2% Agarose Gel (21 min)
1. **Measure 1g of Agarose.**
2. **Prepare Agarose Solution:**
- Mix agarose powder with **49mL of DI water and 1mL of 50x TAE** in a microwavable flask.
3. **Dissolve Agarose:**
- Microwave for **1-3 minutes** until the agarose is completely dissolved.
> **Note:** Do not overboil to avoid buffer evaporation which alters gel percentage. Prefer pulse microwaving to avoid boiling.
4. **Cool Agarose Solution:**
- Let the solution cool down to **about 50°C for about 5 minutes**.
5. **(Optional) Stain Agarose:**
- Add **5uL of SYBR Safe** to the flask.
> **Note:** SYBR Safe binds to DNA, allowing visualization under UV light. If using EtBr, handle it with caution as it's a mutagen.
6. **Pour Gel:**
- Pour the agarose into a gel tray with well comb in place.
7. **Solidify Gel:**
- Place newly poured gel at **4°C for 15 minutes** or let it sit at room temperature for 30 minutes until completely solidified.
> **Note:** Pour slowly to avoid disrupting bubbles. Use a pipette tip to push the bubbles away if they form.
### Loading Samples and Running an Agarose Gel (21 min)
8. **Add Loading Buffer to Samples:**
- Prepare ladder dilution: **Molecular Grade Water + 10uL of 1kb Ladder**.
- Mix at least **10uL of ladder or sample with 2uL of 6x Loading Dye** for a total volume of 12uL.
> For lower DNA concentrations, mix **15uL of ladder or sample with 2.5uL of 6x Loading Dye.**
9. **Place Gel in Box:**
- Once solidified, place the agarose gel into the gel box (electrophoresis unit).
10. **Add Running Buffer:**
- Fill the gel box with **1x TAE** (or TBE) until the gel is covered.
11. **Load Ladder:**
- Carefully load a molecular weight ladder into the first lane of the gel.
12. **Load Samples:**
- Carefully load your samples into the additional wells of the gel.
13. **Run Gel:**
- Run the gel at **80-150V** until the dye line is approximately **75-80% of the way down the gel**.
> Typical run time is about 1-1.5 hours, depending on gel concentration and voltage.
> **Note:** Always run to RED (positive electrode).
### Analyzing Your Gel (21 min)
14. **Stop Run:**
- Turn OFF power, disconnect electrodes, and carefully remove the gel from the box.
> If EtBR was used, place the gel into running buffer and destain.
15. **Visualize Gel:**
- Visualize DNA fragments using a UV light device.
> **Note:** For longer storage or less damage, use long-wavelength UV and minimize exposure time.
**Caution:** When using UV light, protect your hands and eyes by wearing appropriate PPE (Personal Protective Equipment).
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