Methods in Molecular Biology • 16 Enzymes of Molecular Biology

90 Gerard and D'Alessio Ross, J., Aviv, H., Scolnick, E., and Leder, P. (1972) In vitro synthesis of DNA complementary to purif ...

cDNA Synthesis with M-MLV RT 91 Houts, G. E., Miyagi, M., Ellis, C., Beard, D., and Beard, J. M. (1979) Reverse transcriptase f ...

92 Gerard and D'Alessio Helfman, D. M., Fiddes, J. C., and Hanahan, D. (1987) Directional cDNA cloning in plasmid vectors by se ...

cDNA Synthesis with M-MLV RT 93 Saiki, R. K (1989) The design and optimization of the PCR, in PCR Technol- ogy. Principles and ...

CHAPTER 7 Terminal Deoxyribonucleotidyl Transferase (EC 2.7.7.31) Frank Grosse and Andreas Manns Introduction 1.1. The Reaction ...

96 Grosse and Manns research, still nothing is known about the biological function of TdT. Nevertheless, TdT is an important mar ...

Terminal Deoxyribonucleotidyl Transferase 97 sists of 508-529 amino acids. A high degree of sequence homology (>80%) has been ...

98 Grosse and Manns 2(N-morpholino)ethane sulfonic acid (MES) has only one-quarter the activity of TdT in cacodylate. With Tris- ...

Terminal Deoxyribonucleotidyl Transferase 99 i.e., temporarily melted 3' terminus. The melting (or breathing) process of the pri ...

100 Grosse and Manns Experimental Procedures In the following, we will give some examples for the practical use of terminal tra ...

Terminal Deoxyribonucleotidyl Transferase 101 0.5 M Na2HPO 4. Competent cells of E. coli DH5t~ (Gibco-BRL, Eggenstein, Germany) ...

102 Grosse and Manns After measuring the total radioactivity, place the filter disk on a suction device. Rinse several times wi ...

Terminal Deoxyribonucleotidyl Transferase 103 Ethanol-precipitate the remaining DNA in the presence of potassium acetate. 3.2.3 ...

104 Grosse and Manns References Baltimore, D. (1974) Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes? ...

Terminal Deoxyribonucleotidyl Transferase 105 cleotide-polymerizing enzymes of calf thymus gland. I1. Properties of the termi- n ...

CHAPTER 8 Restriction Enzymes Alfred Pingoud, Ji rgen Alves, and Robert Geiger Introduction Restriction enzymes are endonucleas ...

108 Pingoud, Alves, and Geiger Table 1 Classification of Restriction Enzymes Type I Type II Type III Example EcoK EcoRI EcoP Sub ...

Restriction Enzymes 109 tion sequence. Type III enzymes, such as HinfllI (7), are ATP and Mg 2÷ dependent, but do not show a str ...

110 Pingoud, Alves, and Geiger same DNA sequence. They all cleave the DNA within or next to the recognition site producing "stic ...

Restriction Enzymes 111 enzymes. In general, two or three chromatographic steps yield restric- tion enzyme preparations that are ...