Restriction digests of the clone give the following sizes (kb): EcoRI/XhoI--7.4; KpnI--5.8, 1.5; BamHI/XbaI--6.4, 1.0. S. cerevisiae is cotransformed with the lambda clone of interest and vector linearized with an EcoRI/XhoI double digest. Ura+ colonies are tested for complementation of a mutant phenotype. YC-type shuttle vector designed to clone yeast genes that are difficult to clone by rescuing inserts from the vector lambdaMG14 by homologous recombination. The lambdaMG14 fragment closest to the T7 promoter is the same fragment for lambdaMG3 and lambdaMG14. The lambdaMG14 fragment closer to the T3 promoter is a different, slightly larger, MG14-specific fragment constructed by PCR to have the same ends. The order of the major features in the plasmid is: f1 ori - lacZ - T7 promoter - KpnI/lambdaMG14/KpnI - MCS - BamHI/lambdaMG14/XbaI - T3 promoter - pMB1 ori - ampR - CEN6/ARS4 - URA3. |
