How to pour an Agarose Gel

Agarose gels are like Jell-O without the fruit. They have two main ingredients, a buffer and agarose (a highly refined form of agar). We will always use gels that have been made in "0.5X" TBE buffer and our stock solution of TBE is "5X". The percentage gel (w/v) will vary depending on the size of molecules we are trying to resolve. A good starting place is 0.7% (remember that a 1% w/v gel is 1 g agarose in 100 mL buffer). However, our gel molds only hold a volume of 60 mL.

1. Tape the ends of a gel mold and make sure some of the tape wraps around the bottom of the mold by 1 - 2 mm. Choose the appropriate comb(s) and make sure it looks clean.

2. Make 60 mL of the appropriate gel mixture in a 250 mL flask, cover it with Saran, and microwave for 1 minute and 20 seconds on high power (a good starting time).

3. Visually check to see that all the agarose has melted. Unmelted agarose looks like tiny refractive lenses floating around. If not completely melted, nuke it a little longer. Try 20 seconds.

4. Allow the gel to cool a bit; you may hasten this by running cold water over it but do NOT let it cool too much. Don't be startled (and drop the flask) by the popping sound of the saran wrap as the flask cools.

5. Add ethidium bromide (stock EtBr; 10 mg/mL) to the 60 mL of gel so the final concentration is 0.2 µg/mL. Then pour the gel into the mold.
EtBr is a known mutagen so wear gloves.

6. Allow this to cool until it turns slightly white. The gel is ready to run, as soon as you pull off the tape, remove the comb, and submerge it in 0.5X TBE that has the same concentration of EtBr. Unless you are told otherwise, our gel boxes hold 450 mL of buffer.

For 1 L of 5X TBE

0.5 X TBE

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