Sansar
Basic Reagents and Techniques of Molecular Biology
Laboratory Protocols
Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K. Current Protocols in molecular biology. John Wiley & Sons, New York, 1996.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
Basic Tools for DNA Manipulation and Molecular Genetic Analysis
Enzymes Vectors Gel Electrophoresis PCR Southern blotting
Gel Electrophoresis
Gel is a complex net work of polymeric molecules Both DNA and RNA are negatively charged owing to phosphate backbone Nucleic acids migrate in a gel toward anode (+) at a rate that is inversely proportional to the log10 of the molecular weight
Gel Electrophoresis
Migration of nucleic acids depends on pore size (small molecules migrate faster than larger molecules) and configuration of DNA molecules (super coiled molecules > relaxed circles > linear molecules) Use a marker DNA fragments of known size to determine the size of an unknown DNA molecules
Gel Electrophoresis
Agarose Range of separation few hundred to about 20 kb size Agarose in pulsed electric field: for very large fragments Acrylamide Convenient for smaller DNA fragments Polyacrylamide is formed by cross-linking chains of acrylamide with methylene-bis-acrylamide in the presence of Ammonium Persulphate and TEMED (Tetramethyl-ethylene-diamine)
Tracking Dyes
Xylene cyanol
Bromophenol blue
Ethidium Bromide
An intercalating dye As little as 0.05 µg of DNA in one band can be detected as visible fluorescence when the gel is illuminated with ultraviolet light
Agarose Gel Percentages for Resolution of Linear DNA
Gel % 0.5 0.7 1.0 1.2 1.5 2.0 DNA size range (bp) 1,000-30,000 800-12,000 500-10,000 400-7,000 200-3,000 50-2,000
Agarose concentration does not significantly alter the migration of tracking dyes, Xylene cyanol and bromophenol blue, relative to the molecular
Laboratory Protocols
Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K. Current Protocols in molecular biology. John Wiley & Sons, New York, 1996.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
Basic Tools for DNA Manipulation and Molecular Genetic Analysis
Enzymes Vectors Gel Electrophoresis PCR Southern blotting
Gel Electrophoresis
Gel is a complex net work of polymeric molecules Both DNA and RNA are negatively charged owing to phosphate backbone Nucleic acids migrate in a gel toward anode (+) at a rate that is inversely proportional to the log10 of the molecular weight
Gel Electrophoresis
Migration of nucleic acids depends on pore size (small molecules migrate faster than larger molecules) and configuration of DNA molecules (super coiled molecules > relaxed circles > linear molecules) Use a marker DNA fragments of known size to determine the size of an unknown DNA molecules
Gel Electrophoresis
Agarose Range of separation few hundred to about 20 kb size Agarose in pulsed electric field: for very large fragments Acrylamide Convenient for smaller DNA fragments Polyacrylamide is formed by cross-linking chains of acrylamide with methylene-bis-acrylamide in the presence of Ammonium Persulphate and TEMED (Tetramethyl-ethylene-diamine)
Tracking Dyes
Xylene cyanol
Bromophenol blue
Ethidium Bromide
An intercalating dye As little as 0.05 µg of DNA in one band can be detected as visible fluorescence when the gel is illuminated with ultraviolet light
Agarose Gel Percentages for Resolution of Linear DNA
Gel % 0.5 0.7 1.0 1.2 1.5 2.0 DNA size range (bp) 1,000-30,000 800-12,000 500-10,000 400-7,000 200-3,000 50-2,000
Agarose concentration does not significantly alter the migration of tracking dyes, Xylene cyanol and bromophenol blue, relative to the molecular