[Construction of prokaryotic expression vector for MAP30 gene and study of PCR methods for rapid identification of recombinant].
Based on the sequence reported by Lee-Huang,S, we cloned the MAP30 gene of Momordica charantia (balsam pear) into a prokaryotic expression vector pET28a (+). A method by using PCR for rapid identification of positive clone was developed. Result showed this screening method can be used to detect positive colonies from samples of bacterial, purified plasmid, liquid culture,and liquid culture treated with mixture of phenol/Chloroform. The result from liquid-culture-treated- PCR (LCT-PCR) is very close to that of by plasmid-PCR. LCT-PCR is reliable and much easier to used than plasmid-PCR, therefore the LCT-PCR can be used for clone screening during the molecular cloning.
Zhuang
Dong-Hong
DH
Department of Biology, Shantou University, Shantou, Guangdong Province 515 063, China. dhzhuang@stu.edu.cn
Ouyang
Yong-Chang
YC
Hu
Zhong
Z
D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005822:Genetic Vectors; D029777:Momordica charantia / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D054790:Ribosome Inactivating Proteins, Type 2
D017931:DNA Primers; D018744:DNA, Plant; C066347:MAP30 protein, Momordica charantia; D010940:Plant Proteins; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2