Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion

We and others have occasionally experienced difficulty in cloning PCR derived DNA fragments. This has occurred even after extraction of the DNA with phenol and phenol: chloroform, followed by precipitation with ethanol and/or column purification of the bands. To test the hypothesis that the Taq poly...

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Bibliographic Details
Published in:Nucleic acids research 1991-01, Vol.19 (1), p.184-184
Main Authors: Crowe, J.S, Cooper, H.J, Smith, M.A, Sims, M.J, Parker, D, Gewert, D
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
cloning
Cloning, Molecular - methods
DNA-Directed DNA Polymerase - metabolism
Endopeptidase K
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Humans
Immunoglobulins - genetics
Methods. Procedures. Technologies
nucleases
polymerase chain reaction
Polymerase Chain Reaction - methods
proteinases
restriction endonuclease
Serine Endopeptidases - metabolism
Taq Polymerase
Templates, Genetic
Vectors (cloning, transfer, expression). Insertion sequences and transposons
Citations: Items that cite this one
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Summary:We and others have occasionally experienced difficulty in cloning PCR derived DNA fragments. This has occurred even after extraction of the DNA with phenol and phenol: chloroform, followed by precipitation with ethanol and/or column purification of the bands. To test the hypothesis that the Taq polymerase remains bound to the DNA and therefore inhibits restriction endonuclease activity, we incorporated a proteinase K digestion step prior to endonuclease digestion and compared the frequency of cloning of the PCR derived products. We report here an example of the increased cloning efficiency we have achieved after the incorporation of the proteinase K digestion step.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/19.1.184