4 Electrophoresis of DNA and RNA Fragments
73
Polyacrylamide gel electrophoresis (PAGE) is preferred for analysis of
smaller fragments. A 6o/o gel can easily resolve differences of 1 bp in fragments of up to 300 bp in length. Polyacrylamide is usually stored as a 40o/o
stock of the monomerk form mixed with bisacrylamide (a cross-linking
agent) in a ratio of 19:1. TEMED and ammonium persulfate are added
just prior to use to produce polymerization.
Advantages of PAGE:
• It can resolve molecules whose lengths differ by as little as 0.2o/o (i.e., 1 bp
in 500 bp).
• V ery small fragments can be resolved.
• Large quantities of DNA can be separated without loss of resolution: up
to 10 Jlg of a single fragment ofDNA can be loaded in a standard 1 cm x
1 mm slot.
• DNA samples isolated from polyacrylamide gels usually are free of contaminants and ready for use in the most demanding purposes (e.g., microinjection of mouse embryo ).
Disadvantages of PAGE:
• The monomerk form is neurotoxic.
• The gels are a nuisance to prepare and to run.
Agarose is the matrix of choke for evaluating fragments from about 500 bp
up to 10Mb in length. Since DNA fragments of different sizes migrate at
different rates through gels containing different concentrations of agarose,
a concentration is selected whkh allows optimal separation of the range of
sizes desired (Sambrook et al. 1989; Ausubel et al. 1987). Other favorable
aspects of agarase include:
• Gelsare sturdier and can be run in horizontal gel tanks allowing mid-run
evaluation of progress.
• Agarose is non-toxic.
• N ewer preparations of agarose are generally free of inhibitors, and some
(e.g., NuSieve) can resolve fragmentsdown to 10 bp in length.
Hydrolink™. AT Biochemkal (USA) provides a new line of matrkes with an
interesting array of special properties. One matrix (proprietary information
73
Polyacrylamide gel electrophoresis (PAGE) is preferred for analysis of
smaller fragments. A 6o/o gel can easily resolve differences of 1 bp in fragments of up to 300 bp in length. Polyacrylamide is usually stored as a 40o/o
stock of the monomerk form mixed with bisacrylamide (a cross-linking
agent) in a ratio of 19:1. TEMED and ammonium persulfate are added
just prior to use to produce polymerization.
Advantages of PAGE:
• It can resolve molecules whose lengths differ by as little as 0.2o/o (i.e., 1 bp
in 500 bp).
• V ery small fragments can be resolved.
• Large quantities of DNA can be separated without loss of resolution: up
to 10 Jlg of a single fragment ofDNA can be loaded in a standard 1 cm x
1 mm slot.
• DNA samples isolated from polyacrylamide gels usually are free of contaminants and ready for use in the most demanding purposes (e.g., microinjection of mouse embryo ).
Disadvantages of PAGE:
• The monomerk form is neurotoxic.
• The gels are a nuisance to prepare and to run.
Agarose is the matrix of choke for evaluating fragments from about 500 bp
up to 10Mb in length. Since DNA fragments of different sizes migrate at
different rates through gels containing different concentrations of agarose,
a concentration is selected whkh allows optimal separation of the range of
sizes desired (Sambrook et al. 1989; Ausubel et al. 1987). Other favorable
aspects of agarase include:
• Gelsare sturdier and can be run in horizontal gel tanks allowing mid-run
evaluation of progress.
• Agarose is non-toxic.
• N ewer preparations of agarose are generally free of inhibitors, and some
(e.g., NuSieve) can resolve fragmentsdown to 10 bp in length.
Hydrolink™. AT Biochemkal (USA) provides a new line of matrkes with an
interesting array of special properties. One matrix (proprietary information