cDNA Synthesis with M-MLV RT 89
The correction in the numerator takes into account that, on the average,
four nucleotides will be incorporated into the cDNA for every dCTP.
The factor in the denominator is the amount of nucleotide that corre-
sponds to 1 ~g of single-stranded DNA.- Precipitate the product in the remaining diluted sample by adding 0.5 vol
of 7.5M ammonium acetate and 2 vol of cold ethanol, and then centrifug-
ing. Wash the pellet with 70% ethanol, centrifuge again, and dry the pellet
after removing the ethanol. The size of the cDNA product can be analyzed
by alkaline agarose gel electrophoresis (53). Dissolve the sample pellet in
10 I.tL sample buffer (30 mM NaOH, 1 mM EDTA, 10% glycerol, 0.01%
bromophenol blue). Appropriate [32p]DNA size markers should be placed
in sample buffer, e.g., BRL 1-kb DNA Ladder. The gel (1.4% [w/v]) should
be cast in 30 mM NaCI, 2 mM EDTA, and then equilibrated for at least
3 h in alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA)
before loading the samples. For an 11 x 14 cm horizontal gel, electro-
phoresis should be for 5-6 h at 50 V or for 16-18 h at 15 V. Dry the gel
under vacuum, heat for 1-2 h, and expose the gel to X-ray film overnight
at room temperature.
4.4.2. Second-Strand Reaction - Spot duplicate aliquots (10 ~L) from the diluted sample (Section 4.3.2.,
step 5) on separate glass fiber filters, and proceed with determining the
[~-32p]dCTP specific activity and the amount of acid-precipitable radio-
activity as in Section 4.4.1., step 1. The SA of [~-32p]dCTP is given by:
SA (cpm/pmol dCTP) = (cpm/10 ~tL)/(500 pmol dCTP/10 ~L) (3)
The specific activity should be approx 500 cpm/pmol. The amount
of cDNA synthesized in the second-strand reaction is given by:
.Amount of cDNA (lag) = [(cpm) x (50 !11-,/10 ~L) x (150 ~L/10 ]xL)
x (4 pmol dNTP/pmol dCTP)]/[(cpm/pmol dCTP)
x (3030 pmol dNTP/l.tg cDNA)] (3)
Ethanol precipitate the DNA from the remaining diluted sample and
proceed with alkaline agarose gel analysis as described in Section 4.4.1.,
step 2.
References- Baltimore, D. (1970) Viral RNA-dependent DNA polymerase. Nature 226,
1209-1211. - Termin, H. M. and Mizutani, S. (1970) RNA-dependent DNA polymerase in
virions of Rous sarcoma virus. Nature 226, 1211-1213.