pICL

质粒编号:
质粒名称: pICL
其他名称:
质粒大小（bp）: 8400
抗性:
质粒类型: Unspecified
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其他属性: Restriction digests of the clone give the following sizes (kb): KpnI–6.3, 1.5, 0.6; SstI–8.4; BamHI–4.6, 3.8. (ATCC staff) TRP from pYAC4 is not really functional. (personal communication) Linearized vector is transformed into the YAC-containing yeast cells, followed by plating on medium lacking lysine and uracil (and containing 10 mg/L adenine to enhance the red color phenotype of recombinant clones). [1] YAC end fragment clones can be isolated from properly integrated DNA by digestion with one of the enzymes in the polylinker, religation by circularization, and transformation into Escherichia coli. [1] Vector designed to rescue the CEN end of an insert from a yeast artificial chromosome (YAC) constructed in a pYAC4-derived vector. Based on homologous recombination with the pYAC vector. [1] Positive colonies should have the rescue plasmid integrated into the YAC clone near the ScaI site in the beta-lactamase (ampR) gene. [1] DNA from positive colonies can be screened for proper integration of the vector using the following primers: 5'- GCGCTTAATGCGCCGCTACAGGGCG -3' and 5'- GCTCACCGGCTCCAGATTTATCAGC -3', complementary to the f1 ori and ampR sequences respectively. [1] Correctly integrated products will yield a 870 bp amplification product, that should be cleaved by PstI into two fragments: 705 bp and 165 bp. [1] The order of the major features of the rescue vector is: T7 promoter – SacI/polylinker/BamHI – LYS2 – XbaI – SphI – ScaI/ampR – pMB1 ori. [1] The order of the major features in a rcombinant YAC end fragment clone would be: T7 promoter – BamHI/polylinker/SacI – CEN end YAC insert – EcoRI – CEN – ARS – (TRP) – ScaI/ampR – pMB1 ori. [1] Medium is 1227 LB plus ampicillin. Hosts: E.coli, E.coli HB101, E.coli XL-1-Blue, Saccharomyces cerevisiae.
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