Biochemical and Biophysical Research Communications
Limitations on the recombinant plasmid selection by Lac+/Lac− colony phenotype detection
Section snippets
Materials and methods
Enzymes and chemicals. Taq DNA polymerase, T4 DNA ligase, dNTPs, and the kit for DNA isolation were from “MedigenLab” (Novosibirsk, Russia). Restriction endonucleases, T4 polynucleotide kinase, and plasmid pUC19 were from “SibEnzyme” (Novosibirsk, Russia). Plasmids pGEM7Zf and pBluescript IISK were from “Promega” (USA) and Stratagene (USA), respectively. Guanidine thiocyanate, calcium chloride, peptone, yeast extract, and BactoAgar were from “Fluka” (Switzerland). Silica (Silicon dioxide) and o
Results and discussion
A large part of exon 11 of human brca1 gene (2.97 kb, EcoRI/AsuNHI) was cloned in pGEM7Zf plasmid and the resultant plasmid was designated as pGEM7f+ex11. The cloning was designed in-frame with LacZα gene. All colonies of XL-1 Blue E. coli cells with the recombinant plasmid were blue on the indicator plates in spite of the proved 2.97 kb DNA insert. The activity of β-galactosidase in E. coli cells with the recombinant plasmid () did not differ from β-galactosidase activity of the
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