DNA Agarose Gel Electrophoresis - How to Run a DNA Agarose Gel

Biochemistry, Molecular Biology, and Cell Biology Protocols >> DNA Agarose Gel Electrophoresis - How to Run a DNA Agarose Gel

Solutions:

50x TAE
Tris 121g
EDTA 50mL 500mM (pH the EDTA to pH 8.0 with NaOH first before adding)
Acetic acid 28.55mL
Make up to 500mL

Make 1x TAE from 50x with water

Pouring and Running an agarose gel
- wear gloves when handling an agarose gel, as ethidium bromide is very toxic
- throw away all ethidium bromide waste into the plastic bag (including pipet tips that touch the gel when you load samples, the tape for making the gel, etc.)
- it may be necessary to clean the gel apparatus (the comb and the plastic holder) with some water before using them
- for small gels, use 25 mL of 1x TAE
- for large gels, use 50mL of 1x TAE
- generally, 1% agarose (0.25g in 25 mL) will work for most DNA fragments
- for larger fragments, a lower percentage of agarose can be used, and for smaller fragments, a higher percentage of agarose can be used (0.4% to 2%)
- use the flasks designated for agarose gels to make them
- pour in the 1x TAE, and then add the agarose
- microwave for 30 seconds for small gels, and 60 seconds for large gels
- wait for the gel to cool (to speed up the cooling, it is possible to run the flask under cold tap water)
- while the gel is cooling, place tape on both ends of the plastic holder for the gel and also place the comb in
- when the solutions have cooled to about the temperature of your hand, add ethidium bromide (2uL for small gels, 4uL for large gels)
- rinse out the flask with hot water several times
- when the gel is ready to be run, remove the tape, place the gel into the gel box, and then remove the comb (remember to rinse it)
- when running the gels, set to about 100V (unplug the wire in the other apparatus to prevent heating of the solution in the other apparatus); if doing a gel extraction, set the voltage to 80V
- once the gel is ready to be viewed, turn off the voltage, place it into the plastic tray, and take it to the camera

The ethidium bromide is made by adding 50uL of the concentrated ethidium bromide to 450uL of 1x TAE