TY - JOUR
T1 - Analysis and optimization of recombinant DNA joining reactions
AU - Legerski, Randy J.
AU - Robberson, Donald L.
N1 - Funding Information:
We thank Dr Ron Davis for the gift of’ i6Oi. Dr John Wilson for samples of SV40 DNA. and Dr Paul Nodrich for EcoRI endonuclease. We also express our gratitude to Carolyn Peterson and Heidi Eberspaecher for technical assistance, Janie Finch and Janet Nayuin for help in preparation of the manuscript, and Dr Horacze 11. Gray Jr for helpful discussions throughout the course of this work. This investigation was supported by grant G-841 from the Robert A. Welch Foundation and aided by grant no. 1X-121-A from the American Cancer Society and KTH grant no. 5511-17.
PY - 1985/1/20
Y1 - 1985/1/20
N2 - The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mm-Tris · HCl (pH 7.8), 20 mm-dithiothreitol, 10 mm-MgCl2, 1 mm-ATP) at 12 °C to be 1030(± 116) Å. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.
AB - The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mm-Tris · HCl (pH 7.8), 20 mm-dithiothreitol, 10 mm-MgCl2, 1 mm-ATP) at 12 °C to be 1030(± 116) Å. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.
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U2 - 10.1016/0022-2836(85)90093-2
DO - 10.1016/0022-2836(85)90093-2
M3 - Article
C2 - 2984433
AN - SCOPUS:0022429115
SN - 0022-2836
VL - 181
SP - 297
EP - 312
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -