Open AccessAn optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors.
Author(s) -
Z. Topcu
Publication year - 2000
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.2000_4002
Subject(s) - dna ligase , insert (composites) , microbiology and biotechnology , dna polymerase , in vitro recombination , ligase chain reaction , ligation , dna , dna polymerase i , polymerase chain reaction , plasmid , dna clamp , dna polymerase ii , polynucleotide , cloning vector , dna polymerase mu , chemistry , polymerase , biology , recombinant dna , molecular cloning , circular bacterial chromosome , biochemistry , vector (molecular biology) , multiplex polymerase chain reaction , complementary dna , gene , reverse transcriptase , materials science , composite material
This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.
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