Showing papers by "Alan T. Bankier published in 1993"

[13] Preparation and fluorescent sequencing of M13 clones: Microtiter Methods

Abstract: Publisher Summary This chapter discusses various preparation and fluorescent sequencing of M13 clones that is by the microtiter methods. Fluorescent sequencing is found to be the fastest and most efficient way to generate sequence data, when combined with microtiter protocols for template preparation and sequencing reactions. DNA fragments up to 134 kb in size; 21 have been sequenced in a single shotgun, in which the DNA fragment is subcloned in M13 and assembled as one contiguous sequence. The development of a semi-automated microtiter protocol for template preparation overcomes a rate-limiting step in large sequencing projects. The current limitation in the procedure described, using the Applied Biosystems 373A, is gel throughput. However, this method offers significant advantages. Particularly, the manual digitizing of autoradiographic images is no longer necessary, and it consistently obtains greater than 400 nucleotides of accurate sequence per clone. The combination of this base-calling capability with high-throughput microtiter methods for template preparation and sequencing reactions is an effective implementation of the shotgun strategy.