Worm Breeder's Gazette 9(2): 27
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The map has advanced relatively slowly in the last six months, because we have been experimenting with means to preselect clones for fingerprinting. Our preferred technique is to prepare six master nitrocellulose filters (150 mm), carrying about 5,000-10,000 clones each and replicate them onto four sets of nylon filters. The masters are glycerinated and frozen; the copies are processed for hybridization. Probes are made by mixing fragments from (usually ten) unattached clones or clones at the ends of contigs. One set of films is deliberately overexposed, without a screen, so as to make a sharp image of all the colonies to serve as a template. Films from subsequent probings are exposed only enough to show a faint background; they are then aligned with the template films and the positive clones are marked on the latter. With the marked template positioned above its master filter and a suitably positioned lamp, one can align the pattern, sight through the mark to the corresponding master colony and pick correctly more or less every time. This method avoids the need to rescreen (if any clone is picked erroneously it will simply fail to overlap with the probe in subsequent fingerprinting) and so allows a satisfactory throughput. However, we feel that random cloning is still slightly more effective at present and are going back to that for the moment. We are processing clones faster now, as a result of getting a computerized film scanner working and of preparing cosmid DNA in microtitre wells.