Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis

Qilong Xu, Deqing Zhang, Bruce Downie

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (<50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.

Original languageEnglish
Pages (from-to)111-117
Number of pages7
JournalMolecular Biotechnology
Issue number2
StatePublished - Feb 2005


  • Dephosphorylation
  • Digestion
  • Gel electrophoresis
  • Ligation
  • Selection
  • Spin column

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology


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