OF DNA FRAGMENTS
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
tray containing the
still melted agar
This page has been accessed times since 2 October 2001.
16 March 1994, rvsd 27 Feb. '96, 3 Jan. '96, 25 Feb '97, 6 Jan '98, 16 Feb 01
|loading the wells with
prepared DNA samples
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.
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
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.|
|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|
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 is a gel run in 2003. We have experimented with
camera settings to enhance the resolution of weak bands. This was
taken in the black and white mode, and lists the digests of lambda DNA:
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)