8 Pulsed-Field Gel Electrophoresis: Protocols
113
• Buffer concentration
Standard is 0.25-0.5 x TBE; the higher the buffer concentration, the sharper the bands observed. For gel runs which take Ionger than two days, 0.5
x TBE has the advantage of optimal buffering.
• Position of electrodes
Shape of the lanes and sharpness of the bands are affected by the position
ofthe electrodes; in all OFAGE boxes, the angle between point electrodes
should be >90°C.
These protocols are based on lab protocols originally developed in the laboratory of Hans Lehrach. W e wish to gratefully acknowledge Denise Barlow and Margit Burmeister. We thank Jocelyn Faberman for assistance in
preparing the manuscript and RalfKeil for the helpful discussions on PFGE
conditions.
References
Anand R (ed) (1992) Techniques for the analysis of complex genomes. Academic Press,
London, UK
Barlow DP, Lehrach H (1987) Genetics by gel electrophoresis: The impact of pulsed-field
electrophoresis on mammalian genetics. Trends Genet 3:167-171
Bickmore WA, Bird AP (1993) The use of restriction endonucleases to detect and isolate
genes from mammalian cells. Methods Enzymol216:224-244
Bird AP (1986) CpG-rich islands and the function ofDNA methylation. Nature 321:209213
Birren B, LaiE (ed) (1993) Pulsed-field gel electrophoresis, a practical guide. Academic
Press, London, UK
Burmeister M, Ulanovsky L ( ed) ( 1992) Pulsed-field gel electrophoresis, protocols, methods and theories. Methods in molecular biology vol12, Humana Press Inc, Totowa,
NY, USA
Burke DT, Carle GF, Olson MV (1987) Cloning oflarge segments of exogenous DNA into
yeast by means of artificial chromosome vectors. Science 236:806-812
Carle GF, Frank M, Olson MV (1986) Electrophoretic separation oflarge DNA molecules
by periodic inversion of the electric field. Science 232:65-68
Chu G, Vollrath D, Davis RW (1986) Separation oflarge DNA molecules by contourclamped homogenous electric fields. Science 234:1582-1585
Michiels F, Burmeister M, Lehrach H (1987) Derivation of clones close to met by preparative field inversion gel electrophoresis. Science 236:1305-1308
Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed
field gradient gel electrophoresis. Cell 37:67-75
Southern EM, Anand R, Brown WRA, FleteherDS (1987) A model for the separation of
large DNA molecules by crossed field gel electrophoresis. Nucl Acid Res 15:5925-5943
113
• Buffer concentration
Standard is 0.25-0.5 x TBE; the higher the buffer concentration, the sharper the bands observed. For gel runs which take Ionger than two days, 0.5
x TBE has the advantage of optimal buffering.
• Position of electrodes
Shape of the lanes and sharpness of the bands are affected by the position
ofthe electrodes; in all OFAGE boxes, the angle between point electrodes
should be >90°C.
These protocols are based on lab protocols originally developed in the laboratory of Hans Lehrach. W e wish to gratefully acknowledge Denise Barlow and Margit Burmeister. We thank Jocelyn Faberman for assistance in
preparing the manuscript and RalfKeil for the helpful discussions on PFGE
conditions.
References
Anand R (ed) (1992) Techniques for the analysis of complex genomes. Academic Press,
London, UK
Barlow DP, Lehrach H (1987) Genetics by gel electrophoresis: The impact of pulsed-field
electrophoresis on mammalian genetics. Trends Genet 3:167-171
Bickmore WA, Bird AP (1993) The use of restriction endonucleases to detect and isolate
genes from mammalian cells. Methods Enzymol216:224-244
Bird AP (1986) CpG-rich islands and the function ofDNA methylation. Nature 321:209213
Birren B, LaiE (ed) (1993) Pulsed-field gel electrophoresis, a practical guide. Academic
Press, London, UK
Burmeister M, Ulanovsky L ( ed) ( 1992) Pulsed-field gel electrophoresis, protocols, methods and theories. Methods in molecular biology vol12, Humana Press Inc, Totowa,
NY, USA
Burke DT, Carle GF, Olson MV (1987) Cloning oflarge segments of exogenous DNA into
yeast by means of artificial chromosome vectors. Science 236:806-812
Carle GF, Frank M, Olson MV (1986) Electrophoretic separation oflarge DNA molecules
by periodic inversion of the electric field. Science 232:65-68
Chu G, Vollrath D, Davis RW (1986) Separation oflarge DNA molecules by contourclamped homogenous electric fields. Science 234:1582-1585
Michiels F, Burmeister M, Lehrach H (1987) Derivation of clones close to met by preparative field inversion gel electrophoresis. Science 236:1305-1308
Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed
field gradient gel electrophoresis. Cell 37:67-75
Southern EM, Anand R, Brown WRA, FleteherDS (1987) A model for the separation of
large DNA molecules by crossed field gel electrophoresis. Nucl Acid Res 15:5925-5943