BIOL 2030 Lecture Notes - Lecture 18: Agarose Gel Electrophoresis, Restriction Enzyme, Ethidium Bromide
Gel-Electrophoresis and Restriction Endonuclease Mapping
March 7, 2016
What is the purpose of subjecting DNA molecules to agarose gel-electrophoresis?
• To separate DNA molecules based on their molecular mass or size in base pairs
Movement of DNA in an Electric Field:
• At a neutral pH, DNA is negatively charged due to phosphate groups
• The DNA will then move towards the positive (anode) electrode in an electric field
Agarose is an uncharged polysaccharide purified from agar of the seaweed, Agar agar. Red algae
belongs to the family Gelidiaceae *see the pores become smaller
• Sol state (~45C)
• Initial gel (~100C)
• Final gel (ageing)
Preparation of an agarose gel:
1. Gel tray
2. Prepare barriers to retain the agarose
3. Pour molten agarose into tray
4. Insert comb to form walls before agarose solidifies
5. Load DNA samples in individual wells and apply voltage
*shorter molecules will migrate father down the gel
Size-fractionated DNA can be stained with Ethidium Bromide for visulaization
• Uses Kodak Wratten 22A filter
• Gel is placed over a transilluminator filter over UV light
• Bands in DNA fluoresce
Linear DNA molecules migrate at a rate that is inversely porportiona to the log of their molecular
mass (or number of base pairs)
• Smaller DNA migrate more rapidly than larger sized DNA molecules
• This is because the pores in the agarose gel-matrix impedes the larger DNA molecules
Size-Fractionation of DNA during Agarose Gel-Electrophoresis
• Rate of migration of a linear DNA molecule is inversely related to the log of its
molecular mass
• Generation of a standard curve of known molecular mass, DNA molecules can then be
used to extrapolate the molecular mass of an unknown DNA fragment
• This is often the first stage in the characterization of an unknown DNA molecule
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Document Summary
What is the purpose of subjecting dna molecules to agarose gel-electrophoresis: to separate dna molecules based on their molecular mass or size in base pairs. Movement of dna in an electric field: at a neutral ph, dna is negatively charged due to phosphate groups, the dna will then move towards the positive (anode) electrode in an electric field. Agarose is an uncharged polysaccharide purified from agar of the seaweed, agar agar. Red algae belongs to the family gelidiaceae *see the pores become smaller: sol state (~45c) Preparation of an agarose gel: gel tray, prepare barriers to retain the agarose, pour molten agarose into tray, insert comb to form walls before agarose solidifies, load dna samples in individual wells and apply voltage. *shorter molecules will migrate father down the gel. Size-fractionated dna can be stained with ethidium bromide for visulaization: uses kodak wratten 22a filter, gel is placed over a transilluminator filter over uv light, bands in dna fluoresce.
