MOUSE GENOMIC BAC LIBRARY

RPCI-23

About the library

This mouse BAC library RPCI-23 was constructed by Kazutoyo Osoegawa and Minako Tateno [Genome Res.(2000) 10: 116-128] working in Pieter de Jongs' laboratory at the Roswell Park Cancer Institute, Buffalo. Female C57BL/6J mouse kidney and/or brain genomic DNA was isolated and partially digested with a combination of EcoR1 and EcoR1 Methylase. Size selected DNA was cloned into the pBACe3.6 vector (see enclosed map) at the EcoR1 sites and the ligation products transformed into DH10B cells. The average insert size is 197 Kbp. Further information on the library and the vector can be obtained from the web site: http://bacpac.chori.org/

The library consists of approx. 170,000 clones in 480 microtitre plates (384-well format) providing an 11.2-fold genomic coverage. The plate numbers run from 1 to 480. The library is grown in standard LB broth (see e.g. Sambrook-Fritsch-Maniatis: Molecular Cloning, A Laboratory Manual) with chloramphenicol added to a final concentration of 20 mg/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. 10 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 Chloramphenicol (20 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.

User Information:

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, prior to using another probe.

Subsequent publication:

Please acknowledge the originators of the library, Kazutoyo Osoegawa, Minako Tateno and Pieter de Jong, and Geneservice Ltd for providing the library in any publication arising out of using this resource. An example of how a clone (E.g. 271-m19) should be described in a publication or database entry is: RP23-271M19 from the RPCI mouse BAC library 23.

Version 2.0 22^nd November 2004