Preferencje help
Widoczny [Schowaj] Abstrakt
Liczba wyników
2000 | 47 | 3 |
Tytuł artykułu

An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors

Warianty tytułu
Języki publikacji
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 µg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4°C.
Opis fizyczny
  • School of Pharmacy, Ege University, Izmir, Turkey
  • 1. Maunders, M.J (1993) DNA polymerases; in Enzymes of Molecular Biology (Burrell, M.M., ed.) pp.7-30, Humana Press, Totawa, New Jersey.
  • 2. Clark, J.M. (1988) Novel non-template nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nucleic Acids Res. 16, 9677-9686.
  • 3. Ausubel, F.M., Bent, R., Kingston, R.E., Moore, D.D. Seidman, J.G., Smith, J.A. & Struhl, K. (1987) Current Protocols in Molecular Biology, pp. 3.5.1-3.14.4, John Wiley and Sons, New York.
  • 4. Clayton, D.A. (1982) Replication of animal mitochondrial DNA. Cell28, 693-705.
  • 5. Anderson, S., DeBruijin, M.H.L., Coulson, A.R., Eperon, D.J., Sanger, F. & Young, I.G. (1982) Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J. Mol. Biol. 156, 683-717.
  • 6. Innis, M.A., Gelford, D.H., Sninsky, J.J. & White, T.J. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, New York.
  • 7. Protocols and Application Guide (1991) Promega Corporation 217, 41.
  • 8. Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J. & Goodman, H.W. (1977) Rat insulin genes: Construction of plasmids containing the coding sequences. Science 196, 1313-1319,
  • 9. Eckert, K.A. & Kunkel, T.A. (1991) DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl. 1, 17-24.
Typ dokumentu
Identyfikator YADDA
JavaScript jest wyłączony w Twojej przeglądarce internetowej. Włącz go, a następnie odśwież stronę, aby móc w pełni z niej korzystać.