About the library
This library was constructed by Brian Oliver at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) which is part of the National Institutes of Health (NIH) at Bethesda, MD, USA. Poly (A) + RNA was prepared from dissected testes from 1-5 day old y w [67cl] Drosophila melanogaster flies, after removal of the paragonia and inclusion of the ejaculatory ducts and seminal vesicles. The size fractionated cDNA (1-6kb) was directionally cloned in the Stratagene Uni-Zap XR vector using EcoRI and XhoI restriction sites. pBluescript SK (-) is produced after excision. The host strain is E.coli SOLR. The library is grown in standard LB broth (see e.g. Sambrook-Fritsch-Maniatis: Molecular Cloning, A Laboratory Manual) with ampicillin added to a final concentration of 50 µg/ml and 8% glycerol. The stock plates are stored in a freezer at -70°C.
40 microtitre plates (96-well format) have been gridded making a total of 3840 clones. The plate which have been gridded are numbered 3 - 24, 26 - 37, 40 - 42 and 44 - 46. All plate numbers are prefixed bs.
Production of high-density gridded filters
The library has been gridded in a 4x4 array on one Hybond N nylon membrane (Amersham) using a Genomic Solutions Flexys robot. Each clone has been spotted twice.
||Number of clones spotted
||Size of filter
||7,680 (3,840 x 2)
||22 x 22 cm
Clones spotted on the filters were grown overnight at 37°C on LB agar plates containing Ampicillin (50 µg/ml). The filters were subsequently processed by (a) SDS treatment, (b) denaturation and (c) neutralisation, then dried and crosslinked using a Spectronics Corporation XL-1500 UV Crosslinker. The filters are ready for pre-hybridisation in appropriate buffers.
For successful removal of probes, and to enable re-probing, membranes must NEVER be allowed to dry during, or after, hybridisation and washing. Membranes can be stripped to facilitate re-probing by washing sequentially at RT in 0.2M NaOH for 30 min. and 0.1 x SSC / 0.1% SDS / 0.2M Tris-HCl pH 7.5 for 15 min. A gentler method, which extends the number of probings per filter, is to wash the probed membrane after use in a large volume of pre-hybridisation buffer overnight at 65°C, prior to using another probe.
Please acknowledge both the originator of the library and Source BioScience LifeSciences for providing the library, in any publication arising out of using this resource. An example of how a clone (E.g. 32-g10) should be described in a publication is: bs32g10 from the NIH Drosophila cDNA library.