Electrophoresis in 2% agarose gel

Pathogens „ Agarose gel electrophoresis is a method used in biochemistry and

molecular biology to separate DNA or RNA molecules by size. This is
achieved by moving negatively charged nucleic acid molecules through through an

Electrophoresis
agarose matrix with an electric field (electrophoresis).
electrophoresis). Shorter molecules
move faster and migrate further than longer ones.
„ Components of electrophoresis in 2% agarose gel are:
„ Agarose is purified from agar.
agar. Different purities of agarose are commercially
available as are agaroses with different melting properties. High purity low
melt agarose is often used if the DNA is to be extracted from the gel.
„ TBE or Tris/Borate/EDTA
Tris/Borate/EDTA,, is a buffer solution that consists of a mixture
B.K.Kolita Kamal Jinadasa,
Jinadasa, of Tris base,
base, boric acid,
acid, EDTA,
EDTA, and water.
water.
Post Harvest Technology Division, „ Ethidium bromide is an intercalating.
intercalating. When exposed to ultraviolet light,
light, it
NARA, will fluoresce with a red-red-orange color, intensifying almost 20- 20-fold after
binding to DNA.
DNA. Ethidium bromide is a very strong mutagen, mutagen, and may
Colombo-
Colombo-15, possibly be a carcinogen
Sri Lanka. „ Color marker dye containing a low molecular weight dye such as
"bromophenol blue"
blue" (to enable tracking the progress of the electrophoresis)
and glycerol (to make the DNA solution more dense so it will sink sink into the
wells of the gel

Preparation of electrophoresis. Electroforesis

„ DNA marker consists of a plasmid that is digested to completion
with appropriate restriction enzymes to yield DNA bands suitable
for use as molecular weight standards for agarose gel electrophoresis
„ A gel rack
„ A "comb"
„ Power Supply
„ UV lamp or UV lightbox or other method to visualize DNA in the
gel
„ Preparation of a 2% agarose gel:
1. Take 2 g of agarose and inoculate it into 100 ml of TBE.
2. Heat the mix of agarosa and TBE until all agarose is diluted.
3. Add 2.7 µl of Ethidium bromide.
4. Shake the mix until all Ethidium bromide is diluted
5. Empty the gel into the rack
6. Put a comb in the gel
7. Cool the gel about 10 minutes.
8. Load the gel with samples + color marker dye
9. Connect the gel to the power
10. Watch the results with a UV lamp
Gel rack and combs

Load gel Electrophoresis in 2% agarose gel

DNA marker UV lamp

1
Electrophoresis in 2% agarose gel
„ Typical results