Methods in Molecular Biology • 16 Enzymes of Molecular Biology

CHAPTER 10 DNA and RNA Ligases (EC 6.5.1.1, EC 6.5.1.2, and EC 6.5.1.3) Martin J. Maunders Introduction Ligases are a class of ...

214 Maunders In vitro a variety of activities have been detected. These include the joining of hydrogen-bonded cohesive DNA term ...

Ligases 215 The enzyme will not ligate a 3' phosphate group to a 5' hydroxyl, a 3' hydroxyl to a 5' hydroxyl, a 3' dideoxy nucle ...

216 Maunders 2.2.3. Temperature Rates of reaction are very temperature-sensitive, the optimum depend- ing on the lengths of the ...

Ligases 217 range 0.2-1.0 mM cause a slight increase in activity, though higher con- centrations are inhibitory (33). Co 2÷ and ...

218 Maunders Three types of units are defined commonly. First, a Weiss unit (17) catalyzes the exchange of 1 nmol of 32p from in ...

Ligases 219 1-5 mM DTT 2OO lag/mL BSA 50% Glycerol High concentrations of enzyme are very stable at -20°C. If diluted to 500 U ...

220 Maunders After reaction, the enzyme can be inactivated at 65°C for 10 min or by addition of dimethyl dicarbonate to 0.1% and ...

Ligases 221 of the DNA fragment to be linked. The effective concentration of the DNA can be increased by the use of condensing a ...

222 Maunders higher concentrations favor intermolecular reaction (oligomerization and formation of recombinant molecules). Conde ...

Ligases 223 10X Ligation buffer: 200 mM Tris-HC1, pH 7.6 50 mMMgC12 50 mM DTT 500 pg/mL BSA. 10 mM ATP 3.6.2. Protocol Prepar ...

224 Maunders 4.2. Enzymic Reaction 4.2.1. Nucleic Acid Substrate The normal substrate ofRNA ligase is single-stranded RNA, altho ...

Ligases 225 Experimental Procedures 5.1. Uses of RNA Ligase The first obvious use of RNA ligase is in synthesizing oligomers. M ...

226 Maunders The temperature of reaction must be optimized for the specific appli- cation, conditions ranging from 37°C for 30 m ...

Ligases 227 Murray, N. E., Bruce, S. A., and Murray, K. (1979) Molecular cloning of the DNA ligase gene from bacteriophage T4. ...

228 Maunders / 29. 30. Takahashi, M., Yamaguchi, E., and Uchida, T. (1984) Thermophilic DNA ligase. Purification and properties ...

Ligases 229 Wu, R. and Kaiser, A. D. (1968) Structure and base sequence in the cohesive ends of bacteriophage lambda DNA. J. Mo ...

230 Maunders Sugino, A., Snopek, T. J., and Cozzarelli, N. R. (1977) Bacteriophage T4 RNA ligase. Reaction intermediates and in ...

CHAPTER 11 The BAI, 31 Nucleases (EC 3.131) Horace B. Gray, Jr. and Tao Lu Introduction 1.1. Source The extracellular nucleases ...

232 Gray and Lu The BAL 31 nucleases are remarkable for their resistance to inac- tivation and or denaturation in the presence o ...