Construction of mink cosmid library.

High molecular weight DNA was extracted from mink spleen cells according to Maniatis et al. DNA was partially digested by the restriction enzyme Sau 3A and size fractioned by Sucrose gradient centrifugation (10-40% Socrose, 20 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, 1 M NaC1, 26.000 rpm, 20°C, 24 hours). Fractions were analyzed by gelelecrophoresis (0.7% NuSieve agarose (FMC), 1 x TAE buffer). Fractions containing DNA of approximately 50.000 bp. were pooled and subcloned in the cosmid vector Supercos (Stratagene) according to the manufactures procedure. Ligated DNA was packaged in phage particles Gigapack II Gold, Stratagene). The library was transfected into NM554 cells and plated on NZY-plates containing 50 mg/ml Ampicillin, 0.2% Maltose, 10 mM MgSO4). Individual clones were picked at random and processed for mini-prep DNA preparation. The DNA was dot blotted on Hybond-N+ nylon membranes (Amersham) and screened using a gamma-32P-dATP (Amersham) labeled (GT)9 oligonucleotide (Pharmacia) for hybridization (44°C, overnight; 5 x SSC, 2 x Denhardt, 50 mM Tris, pH 7.4, 100 mg/ml tRNA).

All washes were; 15 min., room tempera ture, 20 min., 51°C, 15 min., room tempeerature: 6 x SSC, 0.5% SDS, 0.05% PoP(sodium pyrophosphate) . Filters were exposed for 3-5 days. Positive clones were digested by either RsaI and PvuII in parallel. Digestions were confirmed by gelelectrophoresis (0.8% agarose, 1 x TAE buffer), followed by Southern blotting (Depurinization in 0.2 M HCI; 10 min., Denaturation in 1.5 M Nacl, 0.5 M NaOH; 20 min., followed by transfer using a vacuum blotter from Hybaid and denaturating buffer as transferbuffer). Hybridizations followed the same procedure as described above. The digested cosmid DNA was subcloned into the plasmid vector pCRScribt (Stratagene), transformed into DH5 cells and plated on SOB-plates containing 75 mg/ml Ampicillin, 30 mg/ml x-gal, 150 mM IPTG.

Clones were analyzed by colonilifting to Nitrocellulose membranes (Sartorius). Filters were hybridized ad described. Positive clones were processed for mini-prep DNA preparation and sequenced using Sangers chain termination reaction procedure. The sequencing reaction were loaded on a ALF DNA sequencer and data were collected by the associated software.

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