Gels

How do I Load Gels for Gel Electrophoresis?

I am having a lot of trouble knowing when I am in the well vs too far in, which makes all the PCR products spill out. Anyone have tips for how to load gels without letting anything come out of the well?

Public Comments

1. The wells in the gel should already have a slit in it to make entry easier. Once you feel that there is a little bit of resistance then you're too far in. Usually, it doesn't take much to already have the pipette tip inside the well. So once you penetrate the gel, you should already be able to dispense whatever you wanted into the well.
2. First, you need to steady your hand. One good method is to rest your elbow on the lab bench, then steady the hand with the micropipet by holding your forearm with the other hand. Once your hand is steady, allow the tip of the micropipet to penetrate the surface of the buffer. Hold it directly upright (at a 90 degree angle) over the well. You don't need to go any further into the gel/buffer than just below the buffer line, above the well. Just slowly depress the micropipet - release the sample too fast, and you're likely to get sample contaminating other lanes. The sample is heavier than the buffer and will fall directly into the well. It takes practice. Ask someone in your lab to help. Good luck!
3. Try to look at the gel from different angles or place a peice of coloured paper behind it to be able to see exactly where the wells are. Once you can see them clearly its a lot easier. A steady hand will definatly help. Make sure when u micropipette it in the well you dont realese the button until after it is out of the gel and solution or you will suck it back up and cause it to come out of the well. Try not to be nervous about it, initially it is daunting but once you get rid of the nerves of it it becomes a lot easier. Depending on what type of gel electrophoresis equipment you are using you can get loading guides which can help you to get them in the gel more easier if you like. Good luck!
4. Steady your hand as the other person said. Position your eyes directly above the well. The wells should appear perfectly rectangular. (If they seem squished, your gel is crappy - old or soft or you pulled the comb out too soon - and you're going to have a hard time. Use a thicker comb, if your wells aren't coming out nicely -- it won't affect the result unless you are overloading nucleic acid, or you need very high resolution.) The buffer should immerse the gel completely. If you place the pipet tip above the well pointing into it, you should not even need to be "in" the well at all - the loading buffer makes the sample dense enough that it will sink right into the well if you are above it. Touching the gel with the tip at all is bad practice. If your sample does not sink nicely, make a new batch of loading buffer and make sure you are using enough glycerol. I used to load all my gels "upside down," with the wells on the side nearest the edge of the bench, because it is easier to visualize where the well is without reaching/looking across and getting refraction through the buffer. Just make sure you don't get the leads mixed up if you do this, especially if you have to share a gel box! Also, if you have enough PCR product and you're gel purifying it, you can make the wells wider by putting Scotch tape on the comb so that it connects 2 wells. You may be having an overflow problem, and a bigger well will help.