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

• Authors: Zeki Topcu
• Languages of publication: EN

Abstracts

EN

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.

Keywords

EN

bacterial transformation; PCR; cloning

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2000
• Volume: 47
• Issue: 3
• Pages: 841-846

Dates

published

2000

received

2000-05-29

accepted

2000-06-12

Contributors

author

Zeki Topcu

• Department of Pharmaceutical Biotechnology, School of Pharmacy, Ege University, Izmir, Turkey

References

• 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. Cell 28, 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.