A group of mutant Klenow fragment derivatives, encompassing positions 668. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. mutagenesis to change 14 amino acid residues within this region. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.ĪB - The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Removal of 3-overhangs Second-strand synthesis of cDNA Site-directed DNA mutagenesis using synthetic oligonucleotides Dideoxy DNA sequencing of single- or. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. This problem has been solved You'll get a detailed solution from a subject matter expert that helps you learn core concepts. N2 - The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. With annotated diagrams, explain the reasons the Klenow fragment is better suited for site-directed mutagenesis than DNA polymerase I. T1 - Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I.
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