Methods in Molecular Biology • 16 Enzymes of Molecular Biology

cDNA Synthesis with M-MLV RT 79

Fig. 1. Effect of random hexamer primer concentration on the mass and length of

eDNA synthesized from mRNA by M-MLV RT and M-MLV H- RT. An autoradio-

graph is shown of [32p]cDNA synthesized from a mixture of 0.67 lag each of Ad-2

mRNA, Hela mRNA, and AMV 35S RNA with the indicated amounts of oligo(dT)12_18

or random hexamers. DNA was fractionated on an alkaline 1.4% agarose gel.

2. 
   
   
   
   7. Reaction Conditions
   
   
   
   


3. 7.1. pH and Metal Ion Requirements
   Table 2 gives the monovalent and divalent metal ion requirements
   of M-MLV RT and M-MLV H- RT. The Mg 2+ optima are quite sharp
   at 3 mM. KC1 concentrations between 0 and 100 mM give product
   yields that are 75% or greater of those achieved at the optimum, 75 mM.
   Although the pH activity curve is broad, the yield of full-length prod-
   uct from a long RNA template (7.5 kb) for both enzymes is maximal
   at pH 8.3 at 22°C (at 37°C, pH 8.0).

2.7.2. dNTPs
The KmS of M-MLV RT for dCTP and dATP are 31 and 24 gM,
respectively (17). The Kms of M-MLV H- RT for dNTPs have not been
determined, but are assumed to be similar. In spite of these relatively
low values, high concentrations (>500 mM) of dNTPs in a reaction
mixture result in the most efficient synthesis of full-length cDNAs.