Gel Electrophoresis

Ethidium Bromide (EtBr) is a potent mutagen (and possible carcinogen), and moderately toxic after acute exposure. EtBr can be absorbed through skin, so it is important to avoid any direct contact with the chemical. Always wear nitrile gloves and a lab coat when handling EtBr. Remember when handling anything in the Gel Room to always wear gloves. EtBr is an irritant to the skin, eyes, mouth, and upper respiratory tract. In the event of skin contact with EtBr, immediately wash the affected area with water.

STEP 1. Casting your gel (1.0% agarose)

1. Assemble your gel tray according to the number of samples and the comb size you wish to use. You have the option of setting a full (large), half (small), or micro gel depending on the number of samples you are running.

   Use gel combs with smaller teeth to accommodate more samples on a gel and pour the smallest gel needed. A large gel (up to four combs) can hold up to 100 samples using combs with small teeth and up to 48 samples using combs with large teeth.

2. Remove a bottle of pre-mixed, melted 1% agarose from the incubator.
3. In the fume hood, pour either 75 mLs (for large gel), 50 mLs (for small gel) or 35 mLs (for micro gel) into a clean, small flask.
4. Return the stock bottle of 1% agarose to the incubator.
5. Very carefully add either 4 µL (for large gel), 2.5 µL (for small gel) or 2 µL (for micro gel) of diluted EtBr to your agarose (use only the designated pipette).
6. Be sure to dispose of your EtBr tip in the proper HAZMAT waste container.
7. Immediately close the bottle of EtBr and return it to its box.
8. Swirl the flask to mix the EtBr and slowly pour your agarose aliquot from the flask into your gel tray. (Note: if you are pouring a half gel, watch the agarose to check that it does not leak under the center partition)
9. Immediately scrub and rinse your flask with warm H2O.
10. Leave your gel to set in the fume hood.
11. Turn off the fume hood light.

The fume hood alarm is very sensitive to airflow. Remember to keep the glass windows mostly closed when you are done working. A small opening is needed. If the fume hood alarm goes off, simply press enter on the keypad and check that the windows are not open too much.

STEP 2. Loading your gel

Gel electrophoresis power supplies pose an electrical hazard. Even at 100 volts, they can deliver a lethal shock. Always be mindful of this when operating them.

• Never use a gel box or cover that is cracked or has connectors that appear damaged.
• Never handle the power supply with wet hands while it is plugged in.
• Load your gel and secure the lid to the box before plugging in your power supply.
• Always press the stop button and UNPLUG the power supply before removing the lid.
• If anything gives you cause for concern with the gel box or power supply (for ex, an unusual smell or sound) be sure to turn off the box and tell the lab manager.

1. Transfer gel cast from the fume hood to the sink counter. Remove combs and place them in the sink.
2. Connect a clean gel box to an UNPLUGGED power supply.
3. Remove the gel tray from the cast and place the tray inside the gel box by aligning the center groove of the tray with the notch in the gel box.
4. Add enough 0.5X TBE running buffer to the box such that your gel is submerged.

5. Cut a piece of parafilm and aliquot out 2 µL of loading dye (either blue or orange is available) for each sample. Blue dye (4x): Use for fragments between 500 – 1,000 bps. It will separate out into a purple band (at about 500 bps) and a blue band (at about 4 kb).

   Orange dye (6x): Use for fragments smaller than 500 bps. It has one orange band that migrates at about 100 bp. Always use Orange dye when loading DNA extracts and PCR products for gel excision.

6. Using the pipette, add 3 µL of PCR product (or, 5 µL of DNA extract) to your loading dye. Use clean tips!
7. Load 2 µL of ladder to the first well (use 100 bp ladder for fragments <1,000 bps, mid-range ladder for fragments 1,000-4,000 bps, and the 1 kb ladder for DNA extractions gel).
8. Using the multi-channel or single-channel pipette, load your samples into the gel wells.
9. Cover your gel box with a lid.
10. Using a dry hand, carefully plug in your power supply.
11. Set your voltage at 100 V and run the gel for approximately 25 minutes (for a small gel) or 40 minutes (for a large gel). For DNA extract gels, run at 135 V for 20 minutes.

    The slower and longer you run your gel, the better the resolution will be between bands.

12. Leave your gloves in the gel room by your gel box or by the sink.
13. Store the remaining PCR product in the white refrigerator in the sequencing lab. Each department has its own assigned space. Be sure your products are labeled well.

    Do not store PCR products in the refrigerator/freezer that is in the Gel Room. This unit is for storage of reagents and chemicals only.

The CCG blue dye contains both the blue and purple bands shown below. The CCG orange dye contains only the orange band.