Gel Electrophoresis

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TAE gel (1%) for Gel Electrophoresis Recipe

Reagent	Large gel	Small gel
TAE buffer (fresh)	150 mL	50 mL
SeaKem(R) LE Agarose	1.5 g	0.5 g
Gel Star(50,000x dilution. Can use higher concentrations)	3 uL	1 uL

Prep instructions:

1. Get appropriate sized Erlenmeyer flask (250 mL for large gel or 125 mL for small gel).

2. Add TAE buffer (fresh- from 15L container) and SeaKem LE agarose.

3. Microwave in 20 sec intervals, mixing in between (grab with rubber holder). You want agarose to be fully dissolved (no grains or specks). Be careful to not let the liquid boil over in the microwave, you’ll get a messYOUhave to clean up. Wad up paper towels or take tin foil and cover mouth of flask when you take out of microwave.

4. Cool to touch on bench. Add Gel Star. Mix.

5. Orient the gel plate where the open sides are against the walls (so nothing will pour out) and add in desired comb (there are grooves where the comb will go).

6. 1. Max volume that can put in a well in the gel plate depends on the height of the specific gel and the dimensions of teeth of the chosen comb.

   2. 1. Type of Comb:	Purple Wide Comb	Purple Narrow Comb	Green Wide Comb	Green Narrow Comb
         Max vol. w/ std. gel volume(inuL)	60 x Thickness*	28 x Thickness*	7 x Height x Thickness*	4 x Height x Thickness*

         *Each comb has two sides. One side with teeth of thickness 1mm and one with teeth of thickness 1.5mm

7. Pour the solution into the gel plate and wait until the gel turns opaque. Take out the comb. Then, gently rotate the gel plate by 90 degrees (wells on side where negative terminal [black] will go).

Running the Gel

Make a 1% agarose gel using TAE. Pour fresh TAE over the well until the TAE level is a little above the gel. If doing a gel extraction, use fresh TAE, if not, use the recycled TAE in the container next to the fresh TAE.

Add loading dye to your samples. The dye is 6X concentrated, so if your sample is 50 uL for example, add 10 uL dye and mix well.

Add 2-3 ul of dyed ladder(s) [50bp or 1kb--see figures below] and all cut plasmid DNA to the extra-wide wells in the agarose gel. Do this CAREFULLY and SLOWLY. A good technique is to slowly stick the pipet tip into the well and move it back and forth to verify that it is in the well. Do not go too far down, or you will tear the gel.

Turn the power supply on and set the voltage to the desired amount. Run large gels between 90-120 V and small gels between 60-90 V (running gels at too high of a current will cause the gel to melt/denature). Verify that the gel holder is positioned with wells at negative terminus (black). Press the “run” button. Verify hydrogen bubble production at negative terminus.

Check on the gel approximately every 10 minutes. When DNA fragments are well separated, stop power supply.

Pro Tip: Take a picture of your gel once bands are sufficiently separated for future reference/dissemination

1kb ladder50 bp ladder

Fun fact: Bands that appear red or orange are ssDNA (or RNA). The emission wavelength increases for GelStar bound to single-stranded nucleic acids, and the emission intensity decreases as well.