GENOMIC LIBRARY
By Scott E. Stachel, Department of Molecular and Cell Biology, Division of Biochemistry, and Molecular Biology, 401 Barker Hall, University of California at Berkeley, Berkeley, CA 94720.
I produced a zebrafish genomic library last fall, and now feel that this reagent has been tested enough so that it can be distributed to other laboratories. The specifics of the library follow:
The library was made from genomic DNA obtained from 72 h zebrafish embryos (post-fertilization). The embryos were from commercially-obtained wild-type fish (Ekkwill Tropical Fish Breeders, Gibsonton, FL). The DNA was partially digested with Sau3A to an average size of 20 kb, the first two nucleotides of the Sau3A ends were filled-in, and the DNA was cloned into Xho-1 digested and partially filled-in Lambda Fix II (Stratagene). The ligation was packaged with Gigapack II XL Packaging Extract (Stratagene), which preferentially size selects for 47-51 kb recombinants (18-22 kb inserts). A primary library of 1.8 x 106 clones was amplified in SRB/P2 to a titre of 4 x 109 pfu/ml. As of March 1993 the titre had dropped to 2 x 109 pfu/ml.
The supplied phage stock should be amplified in XL1-Blue/MRA or SRB (Stratagene), or an equivalent strain. Upon arrival, 70ml of DMSO should be added per ml of stock, and the library subsequently stored at -70C and the titre checked.
I have found that the amplified library contains all genes for which I have screened, including three separate retinoic acid receptor genes, goosecoid, noggin, dor3, otx, zhox21, and alpha-tropomyosin. Inserts have been in the 20 kb size range.
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