Pulsed-field gel electrophoresis for long-range restriction mapping.
This unit describes procedures for generating long-range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose-embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high-molecular-weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing.
Gemmill
Robert M
RM
Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, USA.
Bolin
Richard
R
Albertsen
Hans
H
Tomkins
Jeff P
JP
Wing
Rod A
RA
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D015825:Chromosomes, Fungal; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field; D005826:Genetics, Medical; D006801:Humans; D008970:Molecular Weight; D015183:Restriction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D012685:Sepharose
D004247:DNA; D012685:Sepharose