Methods in Molecular Biology • 16 Enzymes of Molecular Biology

10 Weir nicks are opposite. Monitoring of the reaction therefore will not indicate the presence of DNA molecules of intermediate ...

Deoxyribonucleases of Molecular Biology 11 promote the binding of two DNase I molecules at opposite sites on the two DNA strands ...

12 Weir 3.2. Assay of DNase Activity Many methods have been used in the past to assay DNase activity: Melgar and Goldthwaite (2a ...

Deoxyribonucleases of Molecular Biology 13 RNase solution for 15 min (RNase I is a remarkably stable enzyme) and cool to room te ...

14 Weir nuclease inhibitors. RNases are dealt with in much more detail else- where in this volume (Chapter 13). 3.5. "Degradativ ...

Deoxyribonucleases of Molecular Biology 15 technique the DNA of interest is labeled at either the 3' or 5' end and protein is bo ...

16 Weir 2b. Melgar, E. and Goldthwaite, D. A. (1968) Deoxyribonucleic acid nucleases. II. The effects of metals on the mechanism ...

CHAPTER 3 DNA Polymerases (EC 2.7.7.7) Martin J. Maunders Introduction DNA polymerases catalyze the synthesis of DNA molecules ...

18 Maunders The choice of enzyme depends on the reaction conditions employed and the desirability of other inherent enzymatic ac ...

DNA Polymerase 19 DNA Polymerase I (EC 2.7.7.7) DNA polymerase I, Pol I, or Kornberg polymerase is the product of the pol A gen ...

20 Maunders in this manner. DNA polymerase I is the only common DNA poly- merase that can carry out this coupled reaction by vir ...

DNA Polymerase 21 0.5M EDTA, pH 8.0. TNE buffer: 10 mMTris-HCl, pH 8.0 100 mM NaC1 1 mM EDTA TE buffer: 10 mMTris-HC1, pH 8 ...

22 Maunders Klenow Fragment 3.1. Enzyme Data By treatment of DNA polymerase I with the protease subtilisin, a polypeptide of mo ...

DNA Polymerase 23 the labeled probe produced is strand-specific, and there is no produc- tion of hairpin-like artifacts. This re ...

24 Maunders 3.3.1.2. EQUIPMENT REQUIRED 1.5-mL Microfuge tubes. Microfuge. Boiling water bath or dry heating block. Water bath ...

DNA Polymerase 25 3.3.2.3. PROTOCOL Dilute 1 ~tg of target DNA to 44 laL with distilled water. Add 5 IxL of reaction buffer. Op ...

26 Maunders Experimental Procedures T4 DNA polymerase will function in most restriction endonuclease buffers. An alternative ...

DNA Polymerase 27 5.2. Uses ofT7 DNA Polymerase A major use of native or modified T7 DNA polymerase is in DNA sequencing when fr ...

28 Maunders The major distinguishing feature of Taq polymerase is its extreme thermal stability. The enzyme can withstand temper ...

DNA Polymerase 29 Loeb, L. A., Liu P. K., and Fry M. (1986) DNA polymerase ~: Enzymology, function, fidelity and mutagenesis, i ...