Rapid communication: restriction fragment length polymorphisms in the bovine calpastatin gene.

Polymorphisms. BamHI, EcoRI, and TaqI restriction fragment length polymorphisms at the bovine calpastatin locus. Source and Description of Probe. A 2.2-kb cDNA insert coding for Domains 2 through 4 plus a 3’ untranslated region of the bovine calpastatin gene was excised from the pBluescript vector (Stratagene, La Jolla, CA) with EcoRI and XhoI. Method of Detection. Nylon filters were prehybridized in 5x Denhardt’s, .5% SDS with .1 ,ugi,uL of sheared salmon sperm DNA for 2 h at 60°C in a hybridization oven. Random-primed labeled probe (1.0 x los cpm/mL) was added to 10 mL of .5 M NaP04(H3P04), pH 7.4,7% SDS, 1% BSA, .65% PEG and hybridized at 60°C overnight. Filters were washed twice, 15 min each, in prewarmed (60°C) 2x SSC and once in 2x SSC, .l% SDS for 30 min at 60°C. If needed, filters were rewashed in l x SSC, . l% SDS for 15 min at 60°C. Description of Polymorphism. Two codominantly inherited fragments were detected using BamHI with sizes of 9.0 and 5.0 kb plus one monomorphic band at