title: [Construction of T vectors based on Xcm I recognition site and optimization of PCR fragments for ligation].
abstract: OBJECTIVE
  To construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.
  
  
  METHODS
  We firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.
  
  
  RESULTS
  Our results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.
  
  
  CONCLUSIONS
  Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.
authors.forename: Yi-qiao
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Yan-fang
authors.initials: YF
authors.lastname: Zhang
authors.forename: Chao-liang
authors.initials: CL
authors.lastname: Long
authors.forename: Chun-yue
authors.initials: CY
authors.lastname: Li
authors.forename: Xue-hui
authors.initials: XH
authors.lastname: Long
authors.forename: Wen-yu
authors.initials: WY
authors.lastname: Cui
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Hai
authors.initials: H
authors.lastname: Wang
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006657:Histones / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D006657:Histones