Agarose Gel Preparation Instructions

Pre-lab Preparation

Prepare 1X TBE Working Buffer

1. The provided TBE buffer is in a 5X concentrated form and must be diluted using distilled or deionized water prior to use. The volume of 1X TBE buffer needed will vary depending on the volume required for your electrophoresis chambers. Consult the table below to determine the amount of concentrated buffer and water to be used to prepare various volumes of 1X TBE buffer:

 Volume 1X TBE
Buffer Required
Volume 5X Buffer
Concentrate
Volume Distilled or
Deionized Water
100ml 20ml 80ml
250ml 50ml 200ml
500ml 100ml 400ml
750ml 150ml 600ml
1000ml 200ml 800ml
1500ml 300ml 1200ml
2000ml 400ml 1600ml
2500ml 500ml (entire bottle) 2000ml



2. In a large enough container for the volume of 1X TBE buffer being prepared, combine the 5X buffer concentrate and water. Mix thoroughly. Label the container “1X TBE Buffer.”


Prepare 2% Agarose Gels

Note: Always handle hot agarose with heat protective gloves. The activity will require each group to load six samples on an agarose gel. Be sure to use a proper gel comb when pouring the agarose gels.

Microwave Method

1. Set up gel casting trays according to the instructions provided by the manufacturer of your electrophoresis equipment.
2. Loosen the cap on the bottle of prepared agarose.
3. Place the bottle of prepared agarose in a microwave and heat on high for one minute.
4. Wearing heat protective gloves, remove the bottle of prepared agarose from the microwave.
5. Gently swirl the bottle. It may be helpful to squeeze the sides of the bottle after the first interval to break up the prepared agarose a little. This will help the agarose melt faster and more evenly.
6. Repeat, microwaving in one minute intervals, until the prepared agarose is completely melted.

Note: The exact time required to completely melt the agarose will vary by microwave. Simply continue in one minute intervals until the prepared agarose is completely melted. If, during any interval, it appears the agarose is boiling over (coming out of the top of the bottle), stop the microwave and allow the bottle to sit for a few seconds before removing it from the microwave.

7. Pour the liquefied agarose into the gel casting tray. Use the amount of agarose
recommended by the manufacturer of your electrophoresis equipment. Most agarose gels are poured a few millimeters thick.
8. Place a gel comb in the center of the tray containing the agarose. This activity will require wells in the center of the gel.
9. Allow the gel casting tray to sit completely undisturbed until the agarose has
completely solidified (usually approximately 20-30 minutes, depending on the amount of agarose poured).
10. After the agarose has completely solidified, gently remove the gel comb.

Hot Water Bath Method

1. Set up gel casting trays according to the instructions provided by the manufacturer of your electrophoresis equipment.
2. Loosen the cap on the bottle of prepared agarose.
3. Place a glass beaker, taller than the bottle of prepared agarose, on a hot plate.
4. Place the bottle of prepared agarose in the beaker. Add water to the beaker until the level of water is just above the level of agarose in the bottle.
5. Turn the hot plate on and heat until the water in the beaker is boiling.
6. Allow the bottle of agarose to stay in the boiling water until the material in the bottle is completely melted.
7. After the agarose is completely melted, turn off the hot plate and remove the bottle from the beaker wearing heat protective gloves.
8. Pour the liquefied agarose into the gel casting tray. Use the amount of agarose
recommended by the manufacturer of your electrophoresis equipment. Most agarose gels are poured a few millimeters thick.
9. Place a gel comb in the center of the tray containing the agarose. This activity will require wells in the center of the gel.
10. Allow the gel casting tray to sit completely undisturbed until the agarose has
completely solidified (usually approximately 20-30 minute, depending on the amount of agarose poured).
11. After the agarose has completely solidified, gently remove the gel comb.

Copyright 2010 Aldon Corporation