MOUSE GENOMIC PAC LIBRARY RPCI21
About the library
This mouse PAC library RPCI21 provided by the Resource Centre was constructed by Kazutoyo Osoegawa working in Pieter de Jongs' laboratory at the Roswell Park Cancer Institute, Buffalo. (K. Osoegawa et al  Genome research 10: 116-128)
The vector, pPAC4, was constructed by Eirik Frengen (see enclosed map). The source is female 129/SvevTACfBr mouse spleen genomic DNA. The average insert size is 147 Kbp. Further information on the library and the vector can be obtained from the WWW site: http://bacpac.chori.org/home.htm/
The library consists of approx. 128,899 clones in 336 microtitre plates (384-well format). The plate numbers run from 337 to 672 . The library is grown in standard LB broth (see e.g. Sambrook-Fritsch-Maniatis: Molecular Cloning, A Laboratory Manual) with kanamycin added to a final concentration of 25 µg/ml and 7.5% glycerol. The stock plates are stored in a freezer at -70oC.
Production of high-density gridded filters
The library has been gridded in a 4x4 array on 22.2 x 22.2 cm Hybond N nylon membranes (Amersham) using a Genetix Qbot robot. Each clone has been spotted twice to give 36,864 (18,432 x 2) spots on each membrane. 7 filters cover the whole library.
The filters have been labelled as follows:
Clones spotted on the filters were grown overnight at 37oC on LB agar plates containing kanamycin (25 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 65oC, prior to using another probe.
Please acknowledge the originators of the library, Kazutoya Osoegawa and Pieter de Jong, 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. 471-m19) should be described in a publication or database entry is: RP21-471M19 from the RPCI mouse PAC library 21.