LLNL HUMAN SINGLE CHROMOSOME COSMID LIBRARY : Chromosome 19

Chromosome 19
LL19NC02
"F"

About the library

This Chromosome 19 cosmid library was constructed at Lawrence Livermore National Laboratory (LLNL). It has an average insert size of between 32-47 Kb and contains approx. 21,000 clones providing a 9-fold coverage of the chromosome. The vector used is Lawrist 5. The library consists of 55 plates labelled 1- 55. Clones in plates 1-26 are in the E.coli strain ED8767 whilst clones in plates 27-55 are in E.coli strain DH5MCR.

Production of high-density gridded filters

The library has been gridded in a 4x4 array on one large Hybond N nylon membrane, and one small Hybond N nylon membrane (Amersham) using a Flexys robot (Genomic Solutions Ltd). Each clone has been spotted twice.



Clones spotted on the filters were grown overnight at 37°C on LB agar plates containing Kanamycin (20 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.

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 both the originator of the library, LLNL, 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.

Version 1.0 27th March 2001