Agarose Gel Electrophoresis

Objective

Author(s) : Ada Jiang

Determine the size of DNA fragments and/or reaction products based on separation due to size and charge. Following this procedure, gel purification can be conducted to isolate and purify target DNA. This protocol was adapted from Mupid®-One and Thermo Scientific Owl™ EasyCast™ B1A Mini Gel user guides.

Materials

1xTAE buffer
DNA ladder: FroggaBio 1 kB DNA Ladder or NEB 1kb Plus Ladder
DNA loading buffer: Neb Gel Loading Dye, Purple (6X)
SYBR Safe DNA Gel Stain
DNA samples
Agarose powder
Gel electrophoresis equipment

Procedure

Melting agarose solution

Combine 1xTAE and agarose powder to create the desired thickness and concentration of gel for your application.
1. Measure out the agarose powder in a dry 250 mL Erlenmeyer flask
2. Fill the flask with 1xTAE to the needed volume
Microwave for 1:30-2:00 min.

Check every 30s after the initial 1 min, to make sure no over spill due to bubbling
Stop when the liquid has no or minimal visible translucent pellets

Add SYBR Safe into agarose when the liquid stops boiling (<60°C)

10,000 dilution (for example: 60 mL gel needs 6 µL SYBR safe)
Swirl to mix well

Prepare loading samples

Add loading buffer to each sample for a final concentration of 1x dye.
Gently flick the tubes to mix, then spin down.
Consider boiling the samples if enzymes from an upstream reaction could inhibit movement though the gel.

Thermo Scientific™ Owl™ EasyCast™ B1A Mini Gel System

Casting the gel

Insert rubber strips (gaskets) into the grooves of a gel tray, then insert the tray into the buffer chamber so that the gaskets face the walls, sealing off the tray. This is the Casting position.
Insert the correct size comb (10-wells). Carefully pour the molten agarose, and wait until the agarose hardens (~15-20 min).

Use a clean pipette tip to remove bubbles (push them out to the corners).

Once hardened, reorient the entire chamber such that the electrodes are pointing to the right. This is the Running position.
1. Lift the tray up and place it back such that well side is closer to the wall of the chamber that is furthest from your body.
2. If done correctly, the black/negative electrodes will be further from you than the red/positive electrodes.
3. Remove the comb.

Running the gel

Fill up the chamber with 1X TAE buffer (can be reused) until the buffer fully covers the gel.
Load 3 µL of DNA ladder, and add DNA samples. To determine amount of sample to load, estimate the volume of sample needed to load at least 10 ng of DNA or for your intended application.

Application Volume of TAE Max amount of sample
Analysis (thin gel) 30-40 mL 15 µL
Purification (thick gel) 60 mL 50 µL
Run the gel at 100-150V until desired distance.
Prepare a piece of saran wrap large enough to fold over the gel. Retrieve the gel by lifting up the tray and carefully sliding the gel into the saran wrap. Pour the TAE back into its bottle.

Application	Volume of TAE	Max amount of sample
Analysis (thin gel)	30-40 mL	15 µL
Purification (thick gel)	60 mL	50 µL

Mupid®-One Electrophoresis System

Casting the gel

Place the casting stand on a level surface. Insert the center partition if to make a small gel. Insert the gel tray(s).
Insert the correct size comb (13 or 26-wells). Carefully pour the molten agarose, and wait until the agarose hardens (~15-20 min).

Use a clean pipette tip to remove bubbles (push them out to the corners).

Once hardened, transfer the gel trays to the electrophoresis chamber.
1. Remove the comb.
Connect the power supply to the chamber. Running the gel
Fill up the chamber with 1X TAE buffer (can be reused) until the buffer fully covers the gel.
Load 3 µL of DNA ladder, and add DNA samples. To determine amount of sample to load, estimate the volume of sample needed to load at least 10 ng of DNA or for your intended application.

Application Volume of TAE Max amount of sample
13-well (thin gel) 30-40 mL 15 µL
26-well (thick gel) 60 mL 50 µL
Run the gel at 50-135V until desired distance (generally 30 minutes).
Prepare a piece of saran wrap large enough to fold over the gel. Retrieve the gel by lifting up the tray and carefully sliding the gel into the saran wrap. Pour the TAE back into its bottle.

Application	Volume of TAE	Max amount of sample
13-well (thin gel)	30-40 mL	15 µL
26-well (thick gel)	60 mL	50 µL

Visualizing the gel

Check the sample bands using the UV gel imager.
Use upstairs gel imaging devices to visualize the bands and print a copy of the gel.

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