88 Gerard and D'Alessio
....lected to maximize product yield and length. Such labeled cDNA can
be used as a hybridization probe or as starting material for generating a
subtracted cDNA library (38, 39).
In a 1.5-mL tube, dry 10 IlL [t~-32p]dCTP (10 lxCi/~L, >3000 Ci/mmol).
Add on ice 4 ~L 5X First-Strand Buffer, 2 laL 0.1M DTT, 1 Ix[, 10 mM
each of dATP, dGTP, and dTTP, 4 ~L 0.1 mM dCTP, 0.5 ~g mRNA,
0.3 lag pd(N)6, and sufficient water to bring the final vol to 20 laL after
RT is added. Gently vortex the tube, and add 0.5 ~tL (100 U) of M-MLV
H- RT. Incubate at room temperature for 10 min and at 37°C for 1 h.
Add 5 ~L 0.5M EDTA and an equal vol (25 laL) of 0.6N NaOH, and incu-
bate at 68°C for 30 min. Remove unincorporated dNTPs by chromatogra-
phy in 0.1M NaC1, 10 mM Tris-HCl (pH 7.5), and 0.1 mM EDTA over a
Sephadex G-50 column. Recover the product by ethanol precipitation.
Approximately 70% of the total dCTP in the reaction should be incor-
porated, resulting in the synthesis of 1.5 x 108 cpm of labeled product
from 0.5 ~tg of mRNA. Up to 5 lag of RNA can be copied in a 20-pL
reaction. If more than 0.5 lag of RNA is used, the amount of dCTP, [t~-
32p]dCTP, pd(N)6, and RT used should be increased proportionately.
4.4. Product Analysis
4.4.1. First-Strand Reaction
Spot duplicate aliquots (10 ~L) from the diluted sample (Section 4.3.1.,
step 5) on separate glass fiber filters. Dry one filter and count in
scintillant to determine the specific activity of the dCTP in the reaction
mixture. Wash the other filter in TCA-sodium pyrophosphate (Section
4.2.1 .), and dry and count the filter to determine yield. The yield of the
first-strand reaction is calculated from the amount of acid-precipitable
radioactivity determined. In order to perform the yield calculation, the
specific activity of the [{z-32p]dCTP in the reaction must be determined.
The specific activity (SA) is defined as the counts per minute (cpm) of
an aliquot of the reaction mixture divided by the quantity (pmol) of the
same nucleotide in the aliquot.
SA (cpndpmol dCTP) = (cpm/10 IxL)/(200 pmol dCTP/10 laL) (1)
The SA should be approx 200 cprn/pmol. Once the SA is known, the amount
of cDNA in the first-strand reaction can be calculated from the amount of
acid-precipitable radioactivity determined from the washed filter:
Amount of cDNA (lag) = [(cpm) x (50 pL/10 ~L) x (20 ~tL/2 pL)
x (4 pmol dNTP/pmol dCTP)]/[(cprrdpmol dCTP)
x (3030 pmol dNTP/~tg cDNA)] (2)