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 polymerase I. At higher pH, the 5'-3' exonuclease will
attack exposed 5' single-stranded DNA ends more readily; in particular,
the 5' end of a primer-adapter used to prime first-strand cDNA synthe-
sis can be degraded extensively enough so that the restriction endonu-
clease recognition sites are lost. This can lower the output of clones if
the cloning strategy relies on cleavage of one of these sites. Further-
more, the amount of sequence information corresponding to the length of
the 5'-most RNA primer in the reaction normally lost during synthesis
will presumably be minimized by the decrease to pH 7.5 (18). The mini-
mum number of bases lost because of the combined action of RNase
H and DNA polymerase 15'-3' and 3'-5' exonuclease is approx 8 (18).
Finally, T4 DNA polymerase is added at the end of second-strand synthe-
sis to ensure that the termini of the double-stranded cDNA are blunt.
- Experimental Procedures
The following sections refer to conditions optimized for M-MLV
H-RT.
4.1. Laboratory Practices
It cannot be overemphasized that successful cDNA synthesis demands
an RNase-free environment at all times. In general, this will require
the same level of care used to maintain aseptic conditions when work-
ing with microorganisms. In addition, there are several guidelines that
should be followed:
- Never assume that anything is RNase-free except for sterile pipets, cen-
trifuge tubes, culture tubes, and similar labware that is explicitly stated
to be sterile. - Avoid using any recycled bottles and other glassware unless they have
been specifically rendered RNase-free by rinsing with 0.5N NaOH fol-
lowed by copious amounts of sterile, distilled water. Alternatively, glass-
ware can be baked at 150°C for 4 h. - Dedicate laboratory glassware for use with RNA and clearly mark it.
Do not send anything outside of the laboratory to be washed once it has
been rendered RNase free. - Microcentrifuge tubes can generally be taken from an unopened box,
autoclaved, and used for all cDNA work. If necessary, they can be soaked
overnight in a 0.01% aqueous solution of diethylpyrocarbonate (DEPC),
rinsed with autoclaved, distilled water, and then autoclaved.