Electrophoretic Separation of DNA Fragments
NOTE: Latex gloves should be worn at all times to protect from ethidium bromide, and to prevent contamination of DNA samples with skin-borne endonucleases.
Also, prepare a practice gel with many small wells for students to practice loading 15 uL of water with loading dye into them. It makes for much cleaner loading of DNA samples when the “real” gel is run. At the bottom of the page are images of gels taken over the years, good, bad and indifferent…
EQUIPMENT
Ultra-clean glassware:
1 L beaker
1 L graduated cylinder
250 mL beakers
100 mL graduated cylinder horizontal electrophoresis unit
gel tray
power supply
well comb
micropipettors
UV transilluminator
SUPPLIES
latex gloves
TAE buffer (made fr 50x TAE*)
DNA grade agarose
10 mg/mL ethidium bromide
(CAUTION: carcinogen)
masking tape
DNA samples:
undigested lambda DNA
digested lambda DNA (eg: Hind III)
6x loading dye
microcentrifuge tubes
PREPARE THE RUNNING BUFFER AND GEL
1. Prepare running buffer: Dilute 20 mL 50x TAE buffer to 1 liter in dH2O, mix.
2. Prepare the agar : In 250 mL beaker, weight 0.64 g of DNA grade agarose. Add 80 mL 1x TAE buffer, heat to 95 C over flame with stirring (or in microwave 1 min, swirl, microwave for 15 more seconds). Swirl to ensure complete solution.
3. Let cool to 50C.
4. Meanwhile, prepare the tray: tape the ends of the gel tray with masking tape. Press the tape firmly to ensure a good seal on all surfaces.
5. CAUTION, wear gloves: When cooled to 50 C, a dd 4 µL of 10 mg/L ethidium bromide solution to 80 mL agar to make it 0.5 µg/mL.
Click to see the structure of ethidium bromide:
6. Pour agar slab: Place taped tray on a level surface, pour in cooled agar
7. Place the comb at one end.
8. Let sit on level surface until completely solidified
Gently 9. Remove the comb by wiggling and pulling straight up . Do not tear the agar.
10. Remove the tape from the ends.
APPARATUS SET UP, LOADING DNA SAMPLES, RUNNING THE SAMPLES
Set up electrophoresis apparatus: Lie the gel tray with agar in the level electrophoresis apparatus with the wells to the right, towards the black terminals (negative). Fill the apparatus with enough 1x TAE buffer to just cover the gel (filling the wells in the process).
Prepare the DNA samples so that they have 1 to 4 µg of DNA in up to 36 µL of solution containing 1x loading dye (i.e.: 5 µL sample plus 1 µL loading dye, or up to 30 µL sample plus 6 µL loading dye.) Mix either by flicking or by drawing up and down in the micropipet. Load 1 µg of undigested, 4 µg of digested DNA.
loading the wells Load the samples into the wells by loading the pipet with the desired volume of sample: with braced hands, insert the tip into the mouth of the well without touching the sides or bottom, slowly and steadily depress the plunger without shaking or causing bubbles . When the sample is loaded, withdraw the pipet directly out of the well with a smooth movement. The well should have a layer of blue with no irregularities surrounding it. Place standards (undigested DNA or lambda Hind III digest) in the outer wells.
Run the gel: After the wells are loaded
Close the lid, attach the electrodes from the power supply , and turn on the DC power . Set the voltage for 150 volts, and plan to run it for at least an hour , preferably 2 to get the dye 2/3rds across the gel.
HERE ARE GEL IMAGES OF VARIOUS PREDIGESTS TAKEN OVER THE YEARS:
For 100 mL 50x TAE buffer:
24.2 g TRIS (base)
5.71 mL glacial acetic acid
10.0 mL 0.5 M EDTA (14.6 g/100 ml)
