Methods in Molecular Biology • 16 Enzymes of Molecular Biology

cDNA Synthesis with M-MLV RT

Table 1

Comparison of Some Properties of the Commercially Available

RNase H-containing M-MLV RTs

75

Enzyme specific Units used Molar ratio of

Plasmid encoding activity, typically in enzyme to

enzyme Ua/mg b cDNA synthesis mRNA c

pRT601 350,000 200 5:1

pB6B 15.23 35,000 20 5:1

aUnits are determined and defined as described in ref. (20).

bCommercial preparations of both enzymes are nearly homogeneous.

CAssumes an average mRNA is 2 kb in length, and a typical reaction contains 1 lag.

pB6B15.23 RT (35,000-40,000 U/mg of protein) in the same unit
assay (14,19). Since commercial preparations of both proteins are
nearly homogeneous, an equal number of molecules of each enzyme
is used during cDNA synthesis.
M-MLV H- RT is encoded by a modified p601 plasmid and includes
the same first 497 amino acids in cloned M-MLV RT (19), plus five
additional amino acids at the carboxy terminus encoded by the pol
gene read out of frame. The specific activity of M-MLV H- RT is
100,000 U/mg of protein (19).
2.2. Unit Assay
The same unit assay is used by all manufacturers to define the DNA
polymerase activity of RT. Poly(A) • oligo(dT)12_18 is used as the
template-primer, and the unit is defined as nmol of dTMP incorpo-
rated in 10 min at 37°C (20). The unit assay reaction conditions are
not optimal for copying mRNA with either AMV, M-MLV RT, or M-
MLV H- RT.
2.3. Storage
Cloned M-MLV RT and M-MLV H- RT are stable for long periods
of time (>6 mo) when stored at -20°C in a buffer containing 0.1M
NaC1, nonionic detergent, and 50% glycerol. Freezing the enzyme at
-70°C does not affect the unit activity, but does decrease the functional
activity (ability to copy long mRNAs). When stored in 50% glycerol-
containing solutions, the enzymes should never be placed at tempera-
tures at which 50% glycerol freezes.