Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Evaluation of High Throughput M13 DNA Isolation Protocols

Maria de Jesus, Alex Copeland, Jane Lamerdin, Anthony V. Carrano, and Ray Mariella
Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550.

As a preliminary to implementing an automated system for the isolation of DNA from M13 cosmid subclones for our sequencing core facility, we have undertaken an evaluation of several DNA isolation protocols. We describe the results of direct comparisons between four protocols: two commercially available magnetic bead based protocols, a Triton/thermal extraction protocol [1], and a protocol that relies on a PEG precipitation, followed by phenol/chloroform extraction [2].

Currently, we can isolate DNA from approximately 100 subclones per day per person manually using the Qiagen kit and 80 subclones overnight using an Autogen 740 robot. Read lengths of 500-550 bases are obtained on 4.75% polyacrylamide gels with a 34cm well-to-read distance on the ABI 373A DNA Sequencer. Our goal is to increase DNA isolation capacity to approximately 1000 samples per day per person, while maintaining or extending current read lengths.

Template quality and quantity were assayed by agarose gel electrophoresis. Samples were sequenced by dye primer cycle sequencing either manually or using an ABI Catalyst with either Taq Polymerase or Sequitherm (Epicentre Technologies), according to the manufacturers' instructions. Samples were analyzed on 4.75% polyacrylamide gels on an ABI 373A. Protocols were evaluated for sequence quality, read length, robustness, ease of use, automatability, cost, and time.

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-ENG48.

[1] Mardis, E.R., (1994) Nucleic Acids Research, 22(11), 2173.
[2] Zollo, M. and Chen, E., (1994) BioTechniques, 16(3), 370.