The Fugu library provided by the Resource Centre was constructed by Elgar and Nizetic (unpublished data). It has an average insert size of of 40 Kb and consists of approximately 76,000 clones. The vector used is Lawrist 4. More information about the library may be obtained from the Genome News, 20 (1995).
The library has been gridded in a 4x4 array on four 22 x 22 cm Hybond N and one 7.8 x 11.9 cm Hybond N+ nylon membranes (Amersham) using either a PBA Technology Flexys robot or a Genetix Qbot robot. The combination of Hybond N and N+ nylon membranes will not affect subsequent hybridisation if using radioactivity. Each clone has been spotted twice to give 36,864 clones (18,432 x 2) on the large membranes and 2,304 clones (1,152 x 2) on the small membrane. The filters have been labelled as follows:
Label Plates gridded
FUGU1 Plates 1-48 FUGU2 Plates 49-96* FUGU3 Plates101-148 FUGU4 Plates 149-196 FUGU5 Plates 197-199
* plates 97-100 do not exist
Clones spotted on the filters were grown overnight at 37°C on LB agar plates containing Kanamycin (30 mg/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 the originator of the library, Elgar and Nizetic, 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. 171-m19) should be described in a publication is: 171m19 from the Elgar Fugu library.