Electrophoresis II

Introduction

Agarose gel electrophoresis is a method for separating and visualizing DNA fragments produced by restriction digestion of DNA. The fragments are separated by charge and size by forcing them to move through a agarose gel matrix which is subjected to an electric field. The electric field is generated by applying potential (voltage) across an electrolytic solution (buffer). Agarose is a marine colloid purified from algae. When boiled in an aqueous buffer it dissolves, then upon cooling solidifies to a gel. It can be molded into a slab with wells for DNA samples just like jello can be molded into many fun shapes. The pouring of a gel and the set-up of a submarine gel electrophoresis tank is diagramed below. Your instructor will show you the equipment in our lab.

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The agarose solution and running buffer will be prepared by your instructor using these guidelines and buffer components:

Agarose gel:

1% agarose is 1 gram of agarose in 100 mL of 1xTAE.

	For fragments 0.5-5 kb use 1% agarose
	For fragments 0.1-0.5 kb use 1.3 % agarose
	For fragments larger than 5 kb use 0.8% agarose
	For uncut plasmids use 0.8% agarose

1xTAE (Tris/acetate/EDTA) electrophoresis buffer

50´ stock solution, 1 liter:
242 g Tris base
57.1 mL acetic acid
100 mL 0.5 M EDTA, pH 8.0

EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g Na₂EDTA×2H₂O in 700 mL H₂O
Adjust pH to 8.0 with 10 M NaOH (~50 mL): Add H₂O to 1 liter

Protocol

Pour a gel. After the gel has hardened, remove the comb and the dams, and flood the gel with TBE. Mix your DNA samples with DNA loading dye and load them in to the wells on the gel. Also load a molecular weight marker. Connect the leads to the power supply and electrophorese the samples at 70 volts until the dark blue dye reaches the bottom of the gel (1 hour).

The DNA fragments will separate according to size. To see the DNA bands one must stain the gel with ethidium bromide, a fluorescent dye that intercalates DNA, and excite and view the fluorescence by U.V. transillumination. The gel may also be photographed. Unknown bands may be sized by comparison to molecular weight standards. The standards should be used to generate a standard curve for interpolation.

10´ loading buffer
20% Ficoll 400
0.1 M Na₂EDTA, pH 8
1.0% sodium dodecyl sulfate
0.25% Bromphenol blue

Ethidium bromide, 10 mg/ml

Dissolve 0.2 g ethidium bromide in 20 mL H2O

Mix well and store at 4°C in dark

Molecular weight standards profile (make a copy for your note book):

You will be given Lambda Hind III digest or a 1KB ladder as the molecular weight marker.

After you have run the gel, obtain a photograph, and label and measure the migration of the DNA bands. Make a standard curve plot of the known size markers, and determine the size of the unknowns. Construct a table of results for submission with the labeled photograph. The table should include migrations and sizes of all fragments in the study.

Fill out the table for marker fragments,

Results of gel electrophoresis (click to print a copy)

Molecular weight of DNA fragment	Distance migrated (cm)
23,130
9420
6560
4360
2320
2030
560

Fill out the following table for unknown fragments of DNA.

Distance migrated (cm)	Molecular weight (Kb)