Pouring an Agarose Gel

You can make gels in various concentrations. A standard gel is 1%, which is 1g of agarose in 100ml total buffer. For big DNA fragments (>2000kb), you might want to try a 0.8% gel (0.8g agarose/100ml buffer). Our small gel rig holds about 75ml gel total.

We use 10,000x sybrsafe for staining, which calls for 1μl of sybersafe for every 1ml gel. To calculate the amount of stain to add, asses the number of ml of gel total (in our case, 75), move the decimal point to the left one place (i.e. 7.5), and add that many μl of sybersafe to your gel. 

1. In a flask, add 0.75g of agarose and 75ml 1x Lithium Acetate Borate (LAB) buffer 
2. Microwave for 1 minute and 50 seconds. 
3. Check that the agarose looks completely dissolved — the solution should be uniformly transparent. 
4. Add 7.5μl sybersafe, swirl to mix. 
5. Attach the rubber bumpers to the gel tray. 
6. Pour molten agarose into the gel tray, and add the comb. Be sure to orient the comb with the blue bar nearest the edge of the gel so that the wells are formed farthest from that edge. 
7. Let the gel cool for about 20 minutes. It will turn slightly milky when ready, and will feel firm to the touch. 
8. Gently pull the comb straight out without rocking, which may puncture the bottom of the wells. 
9. Gently pull off the rubber pumpers — the one bordering the wells has a tendency to rip the gel along the wells if you are not careful. 

Gels can be made in advance and stored in the refrigerator in a plastic container filled with buffer. They can also be recycled at least once -- after running a gel, you can tear it into strips and stuff the strips into a flask for remelting. When remelting a gel, 1:30 minutes the microwave is generally long enough. Add more sybersafe and cast.