|Two polymorphic microsatellite markers in the European seabass, Dicentrarchus labrax (L.)|
Castilho, R.; McAndrew, B. (1998). Two polymorphic microsatellite markers in the European seabass, Dicentrarchus labrax (L.). Anim. Genet. 29(2): 151
In: Animal Genetics. WILEY-BLACKWELL PUBLISHING, INC: Oxford. ISSN 0268-9146, more
|Authors|| || Top |
- Castilho, R.
- McAndrew, B.
A size-selected genomic library (300-600 bp) was created by partially digesting DNA isolated from seabass muscle with Sau3AI. The DNA was ligated to PUC 18 plasmid (BamHI/BAP, Pharmacia) and transformed into competent E. coli cells (strain DH5; BRL Gibco). Replica nylon filters (Hybond-N-Amersham) were prehybridized (5x SSPE, 5x Denhardt's, 0.5% SDS, 100 mu g ml super(-1) RNA) for 1 h at 65 degree C prior to overnight hybridization at 65 degree C to (GT) sub(15) synthetic oligonucleotides radio-labelled with [ gamma super(32)P]ATP using T4 polynucleotide kinase. Filters were washed twice with 2x SSC, 0.2% SDS at room temperature and with 0.2x SSC, 0 times 2% SDS at 65 degree C, and were exposed to X-ray film (Kodak XAR-5) with two intensifying screens at -80 degree C for 6 h. Positive clones were picked off the plates and grown in LB-ampicillin for 48 h. The plasmid DNA was extracted with phenol-chloroform and minipreparations and followed the classic alkaline lysis. Sequencing was performed with the T7 sequencing Kit (Pharmacia), according to the manufactures instructions, using super(35)S label. Eight per cent acrylamide gels were run for 3 times 5 h at 60 mA. The gel was fixed for an hour and then was dried in a Biorad gel dryer for 2 h before being exposed overnight to Kodak X-omat R film. The sequences of Dla 6 and Dla 11 have been submitted to the GenBank/EMBL database and have accession numbers Y13158 and Y13159, respectively.