Michael Court
Updated September 2006
DNA agarose minigel
1.
Wear gloves since ethidium bromide is mutagen!!!
2.
Use 500 mL pyrex beaker
3.
Weigh out agarose powder (1% gel = 0.6 gram / 60 mL; 1.5% gel = 0.9 gram / 60 mL; 2% gel
= 1.2 gram / 60 mL) in clean weigh boat
a. 1% gel is for DNA > 2kb
b. 1.5% gel is for DNA 1-2 kb
c. 2% gel is for DNA <1kb (or 2-3% could use 3:1 agarose)
4.
Add 1X TAE buffer (or 1X 10 mM Boric acid pH 8.0 for routine PCR electrophoresis) and
mix thoroughly by swirling
5.
Cover top with plastic wrap (2 layers)
a. Punch 5-10 holes in the top to allow venting
6.
Microwave on high until it just starts boiling (about 1 min) – watch carefully!
7.
Swirl to mix contents thoroughly -- VERY HOT!!
a. Use orange hot mitts and hold with both hands
8.
Return to microwave and boil again
9.
Stop when all of agarose is melted
10.
Sit on bench until cooled down
a. generally 5-10 min (when can touch flask without getting burnt)
11.
Add ethidium bromide and mix
a. [2.5 uL of 10 ug/uL solution = 25 ug] per 60 mL gel
12.
Pour into clean gel tray with comb (wash in distilled water beforehand)
13.
Allow to set for 1 hour at room temperature
a. Can keep in refrigerator for up to a week if wrapped in plastic wrap
14.
Dilute samples to >= 9 uL and add 10X DNA loading buffer
a. e.g. 9uL sample+ 1uL loading buffer
15.
Place gel into gel box and add 1X TAE buffer (or 1X Borate buffer) to gel box to the top of the
gel – make sure wells are covered
16.
Add samples and DNA size standard (5-10 uL) to the wells
17.
Run at 100 V for ~30-45 minutes (DNA < 2 kb); 150 V for ~15 min for boric acid buffer
a. For larger DNA, run for longer at lower voltage