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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 weight of DNA fragments.

Polyacrylamide Gel Percentages for Resolution of DNA
Gel % 3.5 5.0 8.0 12.0 15.0 20.0 DNA size range (bp) 100-1,000 75-500 50-400 35-250 20-150 5-100

Dye migration in Polyacrylamide Nondenaturing Gels
Gel (%) Bromophenol blue (bp) Xylene Cyanol (bp)

3.5 5.0 8.0 12.0 15.0 20.0

100 65 45 20 15 12

460 260 160 70 60 45

Dye migration in Polyacrylamide Denaturing Gels
Gel (%)
5.0 6.0 8.0 10.0 20.0

Bromophenol blue (bp) 35 26 19 12 8

Xylene Cyanol (bp) 140 106 75 55 28

Acrylamide Gel percentages for Resolution of Proteins
Gel % 8.0 10.0 12.0 Protein size range (kDa) 40-200 21-100 10-40

Molecular Markers

Primary DNA Metabolizing Enzymes
Restriction Enzymes Ligases Kinases DNA and RNA polymerases

Restriction Endonucleases
Specific and reproducible fragmentation of DNA Most important reagent for recombinant DNA technology Palindromic Cut (but not all enzymes)

Types of Restriction Enzymes
Type
I

Activity
Cleavage & Methylation

ATP
Required

Cleavage site
Random site distant from recognition site (1kb or more distance) Within recognition site

II III

Cleavage only Cleavage & Methylation

Not required Required

Random sites near recognition site e.g. (usually 25 bp away) E.g. EcoP15 recognize two separate non-palindromic sequences that are inversely oriented. Most rDNA work is done with type II Restriction enzymes.

Example of Specificity of Restriction Endonucleases

TaqI HaeIII

Thermus aquaticus Haemophilus aegyptius

T CGA GG CC

Restriction Enzymes

Star Activity
Some restriction enzymes exhibit what are called "relaxed" specificity or "star" activity when they are used under the wrong buffer conditions. For example, EcoRI, which normally only recognizes the sequence G^AATTC, will exhibit EcoRI-star activity if the ionic conditions are too low (e.g. below 50 mM), if the concentration of glycerol is too high, or if too many units of enzyme are added. These...
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