CHICKEN GENOMIC BAC LIBRARY

About the library

This chicken BAC library provided by the Resource Centre was constructed by Richard Crooijmans from Martien Groenen's group at the Wageningen Agricultural University, Department of Animal Sciences, Animal Breeding and Genetics Group, The Netherlands in collaboration with the Texas A&M BAC Centre of the Texas A&M University, College Station, Texas, USA (Crooijmans, RP et al Mamm.Genome [2000] 11:360-363). Work on this resource is co-ordinated by Richard Crooijmans (richard.crooijmans@alg.vf.wau.nl) and by ordering this library you are agreeing to your name being passed on to him.

Female White Leghorn Chicken blood genomic DNA was cloned into the pECBAC1 vector. This vector is a modification of pBeloBAC11 (map, sequence) containing a unique EcoR1 site, the EcoR1 site within the vector having been removed leaving only one EcoR1 site within the lacZ gene (Frijters et al Theor. Appl. Genet. [1997] 94:390-399). The average insert size is 130 kb. Further information on the library and the vector can be obtained from the Chicken Genome Mapping Site

The library consists of approx. 50,000 clones in 130 microtitre plates (384-well format) providing a 5.5-fold genomic coverage. The plate numbers run from 1 to 130. 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 12.5µg/ml and 7.5% glycerol. The stock plates are stored in a freezer at -70°C.

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. 3 filters cover the whole library. The filters have been labelled as follows:

Label Plates gridded

Chicken 1 Plates 1-48

Chicken 2 Plates 49-96

Chicken 3 Plates 97-130

Clones spotted on the filters were grown overnight at 37°C on LB agar plates containing Chloramphenicol (12.5µg/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 at 65°C, prior to using another probe.

Subsequent publication:

Please acknowledge the originators of the library, Richard Crooijmans et al , and Geneservice Ltd. for providing the library in any publication arising out of using this resource. An example of how a clone (E.g. 101-m19) should be described in a publication is: 101m19 from the Chicken BAC library.