User:Anthony Salvagno/Notebook/Research/Notes About Gels
This is a page I should have made a long time ago. It is just random information about gels, gel making, and gel running that I have come across. Other readers' input is also welcomed (see bottom).
Making a gel
Every gel I've made is 1% agarose. This is because the loading buffer has a certain correspondence to DNA sizes. Light blue marker is 3000bp at this percentage for example. You can pour a gel into many different kinds of trays. To make a tray you just simply place autoclave tape at the end of the tray (because there are only 2 walls). You then pour the agarose into your tray and allow to harden. Note: The agarose needs to be relatively cool to pour otherwise it will melt the glue of the tape and the tray will leak. Not fun! You can allow to harden by leaving at room temperature, but this may take up to/more than 20 minutes. I believe you can put the tray into the fridge as well.
After pouring the tray and allowing the gel to harden (form a gel), the tape needs to be removed and the tray can be placed in an electrophoresis device. Fill the device with TAE buffer until the level is over the gel.
You can make different trays (as I mentioned earlier). Each tray needs a certain amount of agarose solution. I've made up to 200mL of agarose for a large tray. I've also needed as little as 30mL for a small gel. In this case you just pour the solution on a glass slide (a big one) and allow surface tension to hold the liquid on the slide. Add too much liquid and the gel will overflow. Gel hardens within 10 minutes.
Prep for a gel
You need to prepare your samples accordingly. Small gels (glass slides) can only hold about 15uL, while larger gels can hold up to 50uL (depending on well size). You need to take that into consideration. You will need:
- Your sample. It needs to be less than the volume you want in the gel to account for addition of other products.
- Loading Buffer. Usually 5x, so you need 1/5th your total volume.
- SDS. We have 10% stock so you will need 1/10th your total volume. This is in case there is protein and enzyme and crap on the DNA so it doesn't mess up the gel.
- Water. Maybe. Sometimes.
Running a gel
This can take a long time. Be prepared for at least an hour (depending on gel length), at high Voltages. This part does allow you to catch up on some internet reading. Good stuff.
Staining
I usually stain 15uL Ethidium Bromide per 150mL TAE buffer. I've heard you can add the Ethidium directly to the gel and simultaneously stain to the running. Staining like normal takes about 30 minutes. After adding the EB to a container of TAE, place the gel (sans tray) in the container, and place on a shaker at low shake.
Can also run a de-stain where you put the gel in water for a long period of time and then take a picture.
