A high throughput system for the preparation of single stranded templates grown in microculture.
A high throughput system for the preparation of single stranded M13 sequencing templates is described. Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein. Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration. Individual glass fiber discs are punched out on the cell harvester and dried briefly. The DNA samples are then eluted in water by centrifugation. The processing time from 96 microcultures to sequence quality templates is approximately 1 hr. Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week. Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987). By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed. Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989). Although minimal human intervention is required, processing time is at least twice as long. Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing. These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here.
Kolner
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DE
Department of Chemistry, University of Wisconsin, Madison 53706-1396.
Guilfoyle
R A
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Smith
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LM
D001483:Base Sequence; D002469:Cell Separation / Q000295:instrumentation; D003090:Coliphages / Q000235:genetics* / Q000254:growth & development; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D004926:Escherichia coli / Q000254:growth & development / Q000821:virology; D005898:Glass; D008969:Molecular Sequence Data; D010472:Perchlorates; D017422:Sequence Analysis, DNA; D017670:Sodium Compounds; D013698:Templates, Genetic; D014776:Virus Cultivation / Q000295:instrumentation
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D010472:Perchlorates; D017670:Sodium Compounds; C031068:sodium perchlorate