DNA gel electrophoresis 1. Wear gloves. Get a gel tray and a comb

DNA gel electrophoresis
1. Wear gloves. Get a gel tray and a comb from the drawer
2. Set the combs into the gel mold, making sure that the comb has the required number
of wells in it.
3. Get an Erlenmeyer flask that can hold at least 4 times the volume you intend to use
(50ml for small gels and 110 ml for big gels).
4. Weigh out the required amount of agarose in a weigh boat or weight paper. This step
depends on the percentage agar you want in your gel. Don’t forget to tare the scale!
Dump the agarose into the flask.
5. Add the right volume of 0.5XTBE buffer, gently swirl to mix. Cover the flask with a
piece of kimiwipe to prevent boiling over (If you need to make more buffer, dilute
5XTBE by 10 times with water in a cylinder. Cover with parafilm and invert a few
times to mix).
6. Microwave 1 minutes. Check and swirl the flask a little. (Be sure to protect your
hand!). Make sure the agarose is completely melted. Add additional time if needed to
melt agarose
7. Cool this flask down in a large beaker filled with tap water, by holding by the neck
and gently swirling. It should be cool enough so that you can hold it in your hand for
some time without it burning you. Or leave the melted agarose in a 50 °C waterbath.
8. Carefully pour the agarose solution into the gel mold and try to avoid bubbles. (Make
sure the comb is in properly in place!) If you do get some bubbles, try to push them
down towards the bottom edge of the gel with a tip.
9. Let the gel set completely and carefully pull up the comb. (submerge the gel in the gel
tank makes it much easier to pull the comb out)
10. Put gel tray inside a gel box. Add enough 0.5XTBE buffer to cover the gel. (for large
gel box, it needs 600-700 ml of buffer)
11. When loading samples, add loading dye. If you have 6X loading dye, you want a 1X
concentration of loading dye in your sample. So, for example, if you have 10
microliters of sample that you want to add dye to, divide it by 5. This gives you 2
microliters of loading dye that you need to add. The total volume of your sample will
then be 12 microliters. So the final concentration of loading dye is 1X. (12 ÷ 12 = 1)
If you have 10X loading dye then add 1 microliter per 10 microliters of sample.
12. When loading samples, it helps to put a dark piece of paper under the gel so that you
can see the wells.
13. Carefully expel your entire sample in the well, avoid going too deep and puncturing
the well and avoid bubbles. If there is some air trapped in your pipette tip, expel it
before loading your sample in the gel. (If an air bubble gets trapped inside the well it
can pop and expel your entire sample).
14. Cover the lid in the right orientation (red matches red, black matches black). Plug the
cord onto the power supply. Make sure to match the plus and minus ends. Adjust the
voltage on the power supply (for large gel, it runs at 100 -120 v, for small gel, it runs
at 90-100 v).
15. When the front dye reaches about 2 cm from the bottom, stop the gel. Stain the gel in
a container with ethidium bromide (EtBr) solution for about 15-20 minutes. View the
DNA under UV. To make ethidium bromide (EtBr) solution, add 5 ul of 10mg/ml
EtBr into a container with about 40ml water. (WARNING: EtBr is a carcinogen,
make sure you wear gloves when handling the material and stained gel. Rinse
thoroughly with water in case you get in contact with a small amount)