Restriction digests of the clone give the following sizes (kb): EcoRI--5.4, 0.48, HindIII--5.8; PstI--6.1; SalI--uncut; BamHI--6.1. Nick-translated insert (sp. act >2x10 E8 dpm/ug) works well with ca. 1-2 x 10 E6 dpm/ml. IMPORTANT: To prevent amplification of a rearranged and/or deleted cosmid, we recommend streaking on LB + amp plates at 30C and picking small colonies for liquid culture. Codominant segregation demonstrated in 20 informative families (63 children) for locus A; for loci B and C, co-dominant segregation demonstrated in 3 families (23 children). Use standard hybridization and wash conditions. Insert gives more consistent results than entire plasmid. 10 E7 dmp/ml of oligolabeled insert (sp act >10E9 dpm/ug) gives clean autorads after 2-5 days exposure. This probe is derived from cosmid 3-3B. |
