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Team:RHIT/Protocols/AgaroseGel
From 2014.igem.org
Agarose Gel Electrophoresis
Agarose Gel Preparation:
.9% agarose gel: add 0.36 g agarose to 40 mL of TBE buffer
Bring to a rolling boil on a hotplate or in a microwave that is not used for food. Once boiling is achieved, remove the mixture and let it cool for 5 min. When cool, pour into the gel box and leave it undisturbed for 20 min. to harden (be sure to place the comb in the gel box before pouring to create wells for samples).
DNA Digest Sample Preparation:
To each sample add the following:
- 10 µL of the digested DNA
- 8 µL of ddH2O
- 1.1 µL of 10X loading dye
Be sure to include a molecular weight ladder in the first lane (2 µL of 1 kb ladder, 1.1 µL of 10X loading dye, and 17 µL ddH2O
Running a Gel:
Once the gel is cool, position it so that the wells in which the samples will be loaded are nearest to the black electrode (DNA particles are negatively charged, and therefore run from the negative electrode to the positive electrode, or from the black end to the red end, when current is applied). Pour TBE buffer into the gel box until both sides are full, and a thin layer of buffer covers the gel. Load the samples in the wells, and record what well each sample is in. Place the top on the gel box, and be sure to place the black end of the top on the black electrode. Turn on the power, and run the gel at around 100 V until the dye is about 75% of the way down the gel. Once the dye reaches this point, turn off the power and disconnect the electrodes. Remove the gel, and place it in EtBr stain (100 mL TBE, 5 µL EtBr) on a rocker for 12-15 minutes. Be cautious, as EtBr is a known mutagen; always wear gloves when working with EtBr. When the gel is done staining, remove it and visualize the bands with any source of UV light. If the gel appears orange in color, you may place it in a small amount of TBE for about 5 min. and de-stain it slightly to remove the orange color.
