74
FENG QIAN AND GREGORY G. GERMINO
Electrophoresis
buffers
Quantity of DNA
loaded
but probably a modified polyacrylamide), called the Lang Ranger, has unique stacking properties that allow more base pairs to be separated per unit
length than conventional acrylamide gels. Still others have features which
allow better separation of molecules with conformational differences (i.e.,
for single-stranded conformation polymorphism analysis). Lastly, there is
another matix which allows clearer resolution of very small DNA fragments
(Hydrolink™).
Generally, the choice of matrix and its concentration depends on the range
of sizes one hopes to resolve.
Of these, TBE (Tris-borate-EDTA) and TAE (Tris-acetate-EDTA) are the
most widely used. Although these buffers have slightly different effects
on DNA mobility and resolution, a buffer is usually selected based on
its relative buffering capacity. TBE has significantly better buffering capacity than TAE and is the buffer of choice for extended pulsed field gel electrophoresis (PFGE) or high-valtage (sequencing gels) electrophoresis. For
historical reasons, TAE is the most commonly used buffer for routine electrophoresis.
The minimum that can be visualized by ethidium staining is about 5 ng. Up
to 200 ng of a single DNA fragment can be loaded into a standard-size weil
(0.5 cm wide by 0.2 cm deep) before over-loading occurs. Over-loading results in trailing, band smearing and reduced mobility. These effects are particularly pronounced with DNA fragments above 10 kb (especially true for
PFGE). The NaCl concentration of a sample has little effect. One advantage
of acrylamide and Hydrolink® gelsisthat much larger quantities of DNA
can be loaded into a lane without causing overloading.
Monitaring electrophoretic separation
Tracking dyes are commonly added to the loading buffer to monitor the
progress of size separation during electrophoresis. The two most widely
used (bromphenol blue and xylene cyanol) have very different electrophoretic mobilities. The fragments with which they co-migrate vary with
the gel matrix and concentration (Sambrook et al. 1989; Ausubel et al.
1987). Two commonly used matrices are
• 1 o/o agarose: xylene cyanol -5 kb, bromphenol blue -0.5 kb;
• 5o/o acrylamide: xylene cyanol -260 bp; bromphenol blue -60 bp.
FENG QIAN AND GREGORY G. GERMINO
Electrophoresis
buffers
Quantity of DNA
loaded
but probably a modified polyacrylamide), called the Lang Ranger, has unique stacking properties that allow more base pairs to be separated per unit
length than conventional acrylamide gels. Still others have features which
allow better separation of molecules with conformational differences (i.e.,
for single-stranded conformation polymorphism analysis). Lastly, there is
another matix which allows clearer resolution of very small DNA fragments
(Hydrolink™).
Generally, the choice of matrix and its concentration depends on the range
of sizes one hopes to resolve.
Of these, TBE (Tris-borate-EDTA) and TAE (Tris-acetate-EDTA) are the
most widely used. Although these buffers have slightly different effects
on DNA mobility and resolution, a buffer is usually selected based on
its relative buffering capacity. TBE has significantly better buffering capacity than TAE and is the buffer of choice for extended pulsed field gel electrophoresis (PFGE) or high-valtage (sequencing gels) electrophoresis. For
historical reasons, TAE is the most commonly used buffer for routine electrophoresis.
The minimum that can be visualized by ethidium staining is about 5 ng. Up
to 200 ng of a single DNA fragment can be loaded into a standard-size weil
(0.5 cm wide by 0.2 cm deep) before over-loading occurs. Over-loading results in trailing, band smearing and reduced mobility. These effects are particularly pronounced with DNA fragments above 10 kb (especially true for
PFGE). The NaCl concentration of a sample has little effect. One advantage
of acrylamide and Hydrolink® gelsisthat much larger quantities of DNA
can be loaded into a lane without causing overloading.
Monitaring electrophoretic separation
Tracking dyes are commonly added to the loading buffer to monitor the
progress of size separation during electrophoresis. The two most widely
used (bromphenol blue and xylene cyanol) have very different electrophoretic mobilities. The fragments with which they co-migrate vary with
the gel matrix and concentration (Sambrook et al. 1989; Ausubel et al.
1987). Two commonly used matrices are
• 1 o/o agarose: xylene cyanol -5 kb, bromphenol blue -0.5 kb;
• 5o/o acrylamide: xylene cyanol -260 bp; bromphenol blue -60 bp.