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! If you have a gel apparatus like this one from Owl, you can pour whatever temperature gel you want into the apparatus and not worry about using tape at all. You can allow to harden by leaving at room temperature, but this may take up to/more than 20 minutes. If you don't have time you can put gel and apparatus in the refrigerator until gel solidifies.
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. The most I have ever cranked my power supply is 140V. The gel gets warm and there is significant bubbles, but I haven't hit the gel melting temperature yet. Even with this voltage I still have had at least 30 min of downtime.
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.
Recently I've been using Sybr Safe gel stain with a blue light transilluminator for visualization. I add the stain to my gel solution (before it gets microwaved) and this works great.
- Ramalldf 03:45, 27 April 2009 (EDT):This is really interesting because I always used to do this in the Osley lab as well. The reason you don't want the voltage to be too high is because there's a chance that you might melt the gel. While I've been working with Alex though I've been a little more free to crank that bad boy up (to about 160 V) without destroying my gel (standard sized, not the large ones you use for Southern blots) and have the gel run in 15-20 minutes. I haven't had time to play around with it too much, but a cool experiment would be to load some ladders and turn it up until you can see when the gel actually is melting. We also use the direct staining method where I add the ethidium to the gel before it finished cooling so that I don't have to stain/rinse afterwards. According to Alex, this doesn't give you as clean looking bands as the alternative method, but for our purposes it has worked well enough.
- Anthony Salvagno 11:25, 27 April 2009 (EDT):Koch told me about the staining method as well. He has some pictures of it, and another downside is that the stain is positively charged, so while the voltage is applied the DNA goes toward the red (positive), but the stain gets pulled toward the black (negative) so longer gels aren't as efficient. As for the time, the power supply I'm using only goes up to 100V so I don't come close to the melting temp based on what you say. That would be an interesting experiment though and we are going to get some gel stuff in KochLab soon, so we could probably run it to see that.