alexa PCR template preparation for capillary DNA sequencing.

Journal of Primatology

Author(s): Silva WA Jr, Costa MC, Valente V, Sousa JF, PaLarson ML, , Silva WA Jr, Costa MC, Valente V, Sousa JF, PaLarson ML,

Abstract Share this page

Abstract Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78\% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85\% of the total number of bases had Phred scores of 15 or above.
This article was published in Biotechniques and referenced in Journal of Primatology

Relevant Expert PPTs

Relevant Speaker PPTs

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords