Pouring Agarose Gels
1. Wear gloves, lab coat, and safety glasses.
2.
Assemble the gel-casting apparatus and insert the plastic comb.
3. Fill a graduated cylinder with 100 mL of water. Pour the water into the gel tray until the water-level is ½
way up the teeth of the comb.
4. Determine how much water was poured into the gel tray. This is the volume of agarose gel you will need
to make. (I usually add five milliliters to this number. It is better to have a few extra milliliters than to not
have enough.)
5. Calculate the mass of agarose you will need to make the desired volume of gel at the desired
concentration. (For example, if you need to make 60 mL of a 2% gel you would multiply .02 X 60.) This will
give you the mass of agarose you will need.
6. Place a weigh boat or weigh paper on the balance and zero the balance. Wait patiently for the balance to
zero. Carefully mass the amount of agarose you will need.
7. Pour the agarose into a 250 mL flask and use the graduated cylinder to add the correct volume of 1X TAE
buffer (not water!) TAE stands for Tris Acetate EDTA.
8. Place a 50 mL flask upside down into the neck of the 250 mL flask to create a reflux chamber. This will
prevent the gel from drying out or boiling over in the microwave.
9. Microwave on 50% power (power level 5) until the reflux chamber starts rattling. Carefully remove from
the microwave and swirl to dissolve the agarose in the buffer. This process of heating until it boils and
then swirling to dissolve usually needs to be repeated several times until all the agarose is dissolved in the
buffer. Hold the flask up to the light or a window and look for undissolved agarose floating in the buffer.
If you can see little swirls catching the light then it isn’t dissolved yet.
10. Agarose that is too hot will crack the gel-casting tray! Let the agarose cool down to about 65 ˚C (until you
can hold the bottom of the flask for a few seconds without burning your hand). If you are adding DNA
stain to the agarose this is the time to add it.
11. Pour the agarose into the gel-casting tray until it ½ way up the teeth of the comb. Remove any bubbles
using a pipet tip. Let solidify for 15-20 minutes.
12. If storing the gel, leave the comb in the gel and leave the gel in the casting tray. Place the tray in a ziplock bag at 4˚C .