Laboratory for Genomics and Bioinformatics

Laboratory for Genomics and Bioinformatics
	Lee H. Pratt	Marie-Michèle Cordonnier-Pratt

		Wet-Lab Pipeline We have established an efficient, low-cost, high-throughput sequencing pipeline. It has been used for sequencing of both complementary and genomic DNA. (click on thumbnail for larger image - 468 kb pdf file)

	After mass excision, colonies are picked with a GeneMachines Mantis robot (1200 colonies/hr in our hands), cultured overnight in freezing medium in 384-well format using a GeneMachines HiGro incubator (48-plate capacity), and stored at -80°C until used.

	Typically, two 384-well plates are thawed and used to inoculate two sets of eight 96-well plates. After overnight culture, the plates are stored at -80°C in two physically separate locations for security. A set of deep-well blocks (1.5 ml/well) is inoculated at the same time. Following overnight growth, plasmids are purified via an inexpensive alkaline lysis protocol. Pipetting is done with a Hydra and all centrifugations are done with a refrigerated plate centrifuge, four blocks at a time.

	Sequencing reactions are performed in 384-well format using 1/12th BigDye reactions. Unincorporated nucleotides are removed with Sephadex G-50 in 384-well plate format and sequencing products lyophilized. Following addition of water, sequencing is performed with an ABI3700. Base calling is done as part of our bioinformatics pipeline.