Insert selection by BamHI methyltransferase protection in P1 phage-based cloning.
Insert selection by BamHI methyltransferase protection in P1 phage-based cloning.
复制标题
P1 噬菌体克隆中通过 BamHI 甲基转移酶保护进行插入选择。
DOI：
10.1016/0378-1119(95)00840-3
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发表时间：
1996
期刊：
影响因子：
3.5
通讯作者：
Wetmur,JG
中科院分区：
文献类型：
--
作者：
Bobovnikova,Y;Kim,SJ;Wetmur,JG
A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 106transformants per μg vector, depending on vector concentration. Cloning of BglII fragments of λ DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per μg vector. Plasmids recovered from these clones were all found to contain a λ BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHI digestion
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