Methods in Molecular Biology • 16 Enzymes of Molecular Biology

70 Cook and Slater Seidman, S., Surzycki, S. J., Delorbe, W., and Gussin, G. N. (1979) Interaction between wheat germ RNA polym ...

Eukaryotic RNA Polymerases 71 Dynan, W. S. and Tjian, R. (1985) Control of eukaryotic messenger RNA syn- thesis by sequence-spe ...

72 Cook and Slater Heintz, N. and Roeder, R. (1982) Transcription of eukaryotic genes in soluble cell-free systems., in Genetic ...

CHAPTER 6 Reverse Transcriptase (EC 2.7.7.49) The Use of Cloned Moloney Murine Leukemia Virus Reverse Transcriptase to Synthesiz ...

74 Gerard and D'Alessio been established for proper handling and storage of the enzyme. Con- ditions for second-strand cDNA synt ...

cDNA Synthesis with M-MLV RT Table 1 Comparison of Some Properties of the Commercially Available RNase H-containing M-MLV RTs 75 ...

76 Gerard and D'Alessio 2.4. Catalytic Properties There are two major difficulties with RT-catalyzed eDNA synthe- sis: (1) mass ...

cDNA Synthesis with M-MLV RT 77 activity as well (7,26). This sets up a competition between the two activities, and the extent t ...

78 Gerard and D'Alessio and M-MLV H- RT are much less sensitive than AMV RT to inhibition by tRNA and rRNA (16; Gerard, G., unpu ...

cDNA Synthesis with M-MLV RT 79 Fig. 1. Effect of random hexamer primer concentration on the mass and length of eDNA synthesized ...

80 Gerard and D'Alessio Table 2 Optima of M-MLV RT and M-MLV H- RT a MgC12, mM % Activity KCI, % Activity pH, H ÷ H- mM H ÷ H- 2 ...

cDNA Synthesis with M-MLV RT 81 The cloned M-MLV RTs are purified free of detectable RNase con- tamination. If precautions are t ...

82 Gerard and D'Alessio Table 3 Comparison of the Reaction Optima of M-MLV RT, M-MLV H- RT, and AMV RT Enzyme Parameter M-MLV RT ...

cDNA Synthesis with M-MLV RT 83 second-strand reaction from 8.3 to 7.5 to suppress the 5'-3' exonuclease activity of DNA polymer ...

84 Gerard and D'Alessio If made with RNase-free labware, most solutions can be made from reagent-grade materials and distilled ...

eDNA Synthesis with M-MLV RT 85 aqueous phase with 2.5 vol of ethanol. Dissolve the tRNA pellet in 0.4M NaCI-0.05M Tris-HCl, and ...

86 Gerard and D'Alessio bring the final vol to 20 gL after RT is added; if second-strand cDNA is to be labeled, omit the [~-32p] ...

cDNA Synthesis with M-MLV RT 87 Add 10 I.tL of 0.5M EDTA to the remaining reaction mixture on ice. The product can now be pheno ...

88 Gerard and D'Alessio . . . . lected to maximize product yield and length. Such labeled cDNA can be used as a hybridization pr ...

cDNA Synthesis with M-MLV RT 89 The correction in the numerator takes into account that, on the average, four nucleotides will b ...