98
FENG QIAN AND GREGORY G. GERMINO
DNA preparation Apreparation ofhigh molecular weight DNA is essential to PFGE analysis.
Usual DNA preparation protocols result in considerable shearing suchthat
the average fragment size is about 100- 200 kb. With special care this range
can be extended up to 400- 500 kb. Consequently, DNA that is to be analyzed by PFGE is generally prepared in agarase blocks, as discussed in other
sections.
Finally, DNA markers ofknown size are as essential to PFGE as for standard electrophoresis. Those in common use include:
• Iambda concatamers (monomer -48 kb): Usual range of resolution is
from -50-1100 kb.
• Saccharomyces cerevisiae: Chromosomes of this yeast range from -200
kb up to 3 Mb (in some strains, the upper Iimit is 1.6 Mb ).
• Candida albicans: Chromosomes range from about 1 Mb to 3 Mb.
• Schizosaccharomyces pombes: Chromosomes range from 3 to 6 Mb.
All of these are now commercially available (Clontech, BRL, Biorad, FMC
BioProducts, etc.). Protocols for preparation of these can be found in Current Protocols in Molecular Biology (Ausubel et al., 1987).
PFGE apparatuses
A number of different apparatuses have been developed over the past few
years:
• OFAGE (Orthogonal Field Agarose Gel Electrophoresis)
In this apparatus, the electrodes are arranged around a square box and
the orientation of the electric field is periodically switched 90 degrees.
One problern with this system is that the electric field within the gel
box is not homogeneous. A consequence of this is that DNA molecules
migrate at different rates depending on their location in the gel.
• FIGE (Field Inversion Gel Electrophoresis)
Size separation can also be produced by simply periodically inverting a
uniform field 180 degrees (Carle et al. 1986). There are several advantages
to FIGE:
- The lanes are straight, allowing accurate lane-to-lane comparisons
and size estimations. This is especially advantageaus for preparative
gels.
FENG QIAN AND GREGORY G. GERMINO
DNA preparation Apreparation ofhigh molecular weight DNA is essential to PFGE analysis.
Usual DNA preparation protocols result in considerable shearing suchthat
the average fragment size is about 100- 200 kb. With special care this range
can be extended up to 400- 500 kb. Consequently, DNA that is to be analyzed by PFGE is generally prepared in agarase blocks, as discussed in other
sections.
Finally, DNA markers ofknown size are as essential to PFGE as for standard electrophoresis. Those in common use include:
• Iambda concatamers (monomer -48 kb): Usual range of resolution is
from -50-1100 kb.
• Saccharomyces cerevisiae: Chromosomes of this yeast range from -200
kb up to 3 Mb (in some strains, the upper Iimit is 1.6 Mb ).
• Candida albicans: Chromosomes range from about 1 Mb to 3 Mb.
• Schizosaccharomyces pombes: Chromosomes range from 3 to 6 Mb.
All of these are now commercially available (Clontech, BRL, Biorad, FMC
BioProducts, etc.). Protocols for preparation of these can be found in Current Protocols in Molecular Biology (Ausubel et al., 1987).
PFGE apparatuses
A number of different apparatuses have been developed over the past few
years:
• OFAGE (Orthogonal Field Agarose Gel Electrophoresis)
In this apparatus, the electrodes are arranged around a square box and
the orientation of the electric field is periodically switched 90 degrees.
One problern with this system is that the electric field within the gel
box is not homogeneous. A consequence of this is that DNA molecules
migrate at different rates depending on their location in the gel.
• FIGE (Field Inversion Gel Electrophoresis)
Size separation can also be produced by simply periodically inverting a
uniform field 180 degrees (Carle et al. 1986). There are several advantages
to FIGE:
- The lanes are straight, allowing accurate lane-to-lane comparisons
and size estimations. This is especially advantageaus for preparative
gels.