Restriction digests of the clone give the following sizes (kb): EcoRI--5.3; BamHI--5.3; PstI--4.30, 0.8. Cosmid clones contain either 3.2 or 2.7 kb of the original vector depending on which cos site was used in packaging. This does not affect the use of NotI sites in restriction mapping of the clone. 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. Cosmid vector with a cloning capacity of 35-45 kb, 3 cos sites, and NotI sites flanking the polylinker sites for ease of restriction mapping. PATENT PENDING. cos4 was digested with BamHI+AvaI, the 1.1 kb fragment removed and the remaining ends filled in. Synthetic NotI linkers were inserted into the EcoRI & HindIII sites. The small fragment between EcoRI & HindIII was replaced with the pUC18 polylinker. |
