Restriction digests analyzed on agarose gels give the following sizes (kb): HindIII--5.4; BamHI--5.4; PstI/HindIII--4.6, 0.8; PstI/SalI--3.3, 2.3. 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. A cosmid vector containing a single cos site. Cloning into the BamHI or EcoRI site involves 2 preparations of dephosphorylated vector, one linearized with HindIII, the other linearized with SalI. An equimolar ratio of right-hand and left-hand cos containing vector fragments, digested with BamHI or EcoRI can be ligated to appropriately digested and dephosphorylated inserts. Only molecules with 2 cos sites separated by 37-50 kb can be packaged. |
