Rapid restriction map constructions using a modified pWE15 cosmid vector and a robotic workstation.
This paper describes a number of techniques for rapid restriction mapping of cosmid clones. First, we have replaced the cloning site of cosmid vector pWE15 with a polylinker containing 15 infrequently cleaved restriction enzyme sites that are placed asymmetrically on each side of the BamHI cloning site. DNA cloned into this vector can be fully recovered by using several pairs of restriction enzymes. Second, we have designed a simple electrical circuit device that allows the performance of asymmetric voltage gradient field inversion gel electrophoresis (AFIGE) to improve the resolution of DNA molecules in the range of 20-50 kbp. AFIGE can be obtained by simply placing the device in between a commercially available switching unit and the gel box in a standard field inversion system. Finally, the restriction digestion procedure has been automated by using a Beckman Biomek 1000 robotic workstation. Using this automated system, 96 restriction reactions, including gel loading, can be performed in less than two hours. In summary, these methods represent at least a tenfold improvement in the speed and/or mapping data that can be obtained in a single gel.
Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
E
E
Lai
K
K
Wang
N
N
Avdalovic
L
L
Hood
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA; D004586:Electrophoresis / Q000379:methods; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012371:Robotics
D004247:DNA