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# Agarose Gel Electrophoresis, 1.2% with Ethidium Bromide

### Goal/Experiment:
The goal of this experiment is to separate DNA molecules based on size using agarose gel electrophoresis. This protocol is suitable for checking DNA after a restriction digest and can resolve DNA fragments ranging from 400bp to 7kb.

### Abstract
This protocol separates molecules based on size and is great for checking DNA after a restriction digest. The protocol utilizes a 1.2% agarose gel, sufficient for resolving DNA from 400bp to 7kb. The electrophoresis system has a 7.5cm x 10cm gel bed area with an approximately 15cm electrode distance. The minimum volume for a 7.5cm x 10cm x 0.5cm gel is 37.5mL, and the maximum volume for a 50mL gel is 0.67cm in height.

**Citation**: Harold Bien Agarose gel electrophoresis, 1.2% with Ethidium bromide. protocols.io dx.doi.org/10.17504/protocols.io.ds76hm  
**Published**: 11 Sep 2015

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### Before Start
- Ensure you have a DNA sample ready, either from PCR or a recently performed restriction digest.
- Dilute the 50X TAE Buffer to 1X using sterile filtered water.
- For the dual-sided 1.0mm gel comb MHC-10-0816 the maximum volume is 35/13μL.
- For the dual-sided 1.5mm gel comb MHC-15-1014, the maximum sample volume is 38/25μL.

---

### Materials

| Reagent/Equipment                                     | Specification                   | Vendor                                           |
|-------------------------------------------------------|---------------------------------|--------------------------------------------------|
| 2-Log DNA Ladder                                      | 100-200 gel lanes               | N3200S by New England Biolabs                    |
| Gel Loading Dye, Purple (6X), no SDS                  | 4.0 mL                          | B7025S by New England Biolabs                    |
| GeneMate LE Quick Dissolve Agarose                    | 500g                            | E-3119-500 by Bioexpress                         |
| Ethidium bromide                                      | 10mg/mL, 10mL                   | X328-10ML by Amresco                             |
| TAE (Tris-Acetate-EDTA) Buffer, 50X                   |                                 | K915 by Amresco                                  |
| Horizontal mini-gel kit                               |                                 | MHU-202 by C.b.s Scientific                      |

---

### Protocol

#### Prep Work

##### Step 1
Weigh out 0.6 g (1.2% w/v of 50mL) agarose and add it to the Erlenmeyer flask.

- **Amount:** 1 g
- **Reagents:** GeneMate LE Quick Dissolve Agarose, 500g [E-3119-500 by Bioexpress]

##### Step 2
Add 50mL of 1x TAE buffer.

- **Amount:** 50 mL
- **Reagents:** TAE (Tris-Acetate-EDTA) Buffer, 1X

##### Step 3
Place Erlenmeyer flask in microwave. Set to wait 30 seconds, then full power (P10, 1250W) for 20 seconds followed by low power (P1, 125W) for 30 seconds or until the solution is clear and agarose is completely dissolved.

**Duration:** 50 seconds

**Notes:**  
- Ensure the agarose is fully dissolved in the buffer solution.

##### Step 4
Remove Erlenmeyer flask from microwave and let it sit on the lab bench to cool just until you can comfortably pick it up.

**Duration:** 3 minutes

##### Step 5
Add 1μL concentrated ethidium bromide (10mg/mL) into the flask and swirl to mix, taking care not to introduce bubbles.

- **Amount:** 1 μL
- **Reagents:** Ethidium bromide, 10mg/mL, 10mL [X328-10ML by Amresco]
- **Notes:** The final concentration of ethidium bromide will be 10μg/50mL or 0.2μg/mL (Carcinogen, handle with care).

##### Step 6
Place gel tray on clamp and clamp securely. Add well plates where you want wells and use a level to ensure it is balanced.

**Notes:**  
- Good well balance is crucial for even sample loading.

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### Running the Agarose Gel

#### Running the Gel

##### Step 7
Pour contents of the Erlenmeyer Flask into the gel tray and let it sit for 30 minutes, or until a blue tint appears.

- **Duration:** 30 minutes

##### Step 8
Remove the well plates carefully to avoid tearing the gel and remove the tray from the clamp, ensuring the gel remains in the tray.

##### Step 9
Place gel tray into the gel electrophoresis apparatus with the wells closer to the negative/black end.

##### Step 10
Pour additional TAE Buffer to fill each side of the apparatus and to create a thin layer of buffer covering the top of the gel.

##### Step 11
Prepare DNA ladder and samples by adding 6x blue dye.

- **Reagents:** Gel Loading Dye Blue (6X) - 4.0 ml [B7021S by New England Biolabs]
- **Notes:**  
  - Dilute the DNA ladder 1:10 in sterile filtered water when using the 1.5mm thick 14-well lane.

##### Step 12
Pipette your samples into each well.

- **Notes:**  
  - For the 1.5mm gel comb MHC-15-1014, recommended DNA mass is 200-500ng for 14-well.

##### Step 13
Place the lid on the apparatus and plug cables into the high voltage power supply. Run at 100V (6.6V/cm) for 45-60 minutes or until the loading dye has sufficiently migrated down the gel.

- **Duration:** 45-60 minutes
- **Notes:**  
  - Ensure that the negative terminal (typically black) is plugged into the negative/black terminal on the power supply and the wells are at the negative side.

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#### Visualizing DNA Bands

##### Step 14
Gel can be imaged on UV transilluminator through the UV-transparent gel tray or removed and wrapped in plastic wrap for storage at 4°C for later use.

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### Warnings
Ethidium Bromide potentially acts as a mutagen or carcinogen. Handle with proper safety measures.

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