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.

Automated Pooling of Large DNA Libraries

Donald C. Uber
Human Genome Center and Engineering Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720

Our mapping strategies require screening large libraries (e.g. 100,000 clones) with multiple probes to find the occurrences of specific DNA sequences. Since the expected number of hits in a library with a particular probe is relatively few (e.g. five), the effort in this task can be greatly reduced by first combining the clones into pools according to a prescribed pattern, and screening just the pools. By observing which pools "light up", it is then straightforward to identify which clones match the probe.

Using a Hewlett-Packard ORCA robot, we have automated a 3D pooling strategy in which 960 clones in 96-well format are combined into 30 pools: 8 row pools, 12 column pools, and 10 plate pools. This scheme reduces the number of screening assays by a factor of 32. The ORCA uses a custom 12-channel pipet tool with standard 9 mm spacing, and makes pools in troughs that accept 12 samples simultaneously. A single disposable pipet tip per clone is used, as compared to three tips when the process is done manually. A run of 10 source plates is completed in less than three hours, including making a copy of each source plate, as compared to five hours manually.