LANL HUMAN SINGLE CHROMOSOME COSMID LIBRARY

Chromosome 5
LA05NC01
"LA05"

About the library

This Chromosome 5 cosmid library was constructed at Los Alamos National Laboratory according to the method described in Longmire, JL. et al Genetic Analysis, Techniques and Applications, 1993, 10 : 69-76. It has an average insert size of 30 - 40 Kb and contains approx. 25,200 clones providing a 4-fold coverage of the chromosome. The vector used is sCOS1, also called superCos1, in E. coli strain DH5a mcr. The library consists of 66 plates labelled 1- 66.

The library has been gridded in a 4x4 array on two Hybond N nylon membranes (Amersham). Each clone has been spotted twice.

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 both the originator of the library, LANL as described in the Materials Transfer Agreement that you have signed, and Geneservice for providing the library, in any publication arising out of using this resource. An example of how a clone (E.g. CLONE NAME = 32-B9) should be described in a publication is: LA0532b9 from the LA05NC01 library.