86 Gerard and D'Alessio
bring the final vol to 20 gL after RT is added; if second-strand cDNA is
to be labeled, omit the [~-32p]dCTP.- If pd(T)12_18 or primer-adapter is present, equilibrate the tube to 37°C
before adding 200-1000 U (1-5 pL) of M-MLV H- RT (SuperScript TM
RT, use 200 U/lag mRNA); if pd(N)6 is used, immediately add the appro-
priate amount of RT, and incubate at room temperature for 10 min;
finally incubate all reaction types at 37°C for 1 h. - The final reaction composition is 50 mM Tris-HCl (pH 8.3),75 mM
KC1, 3 mM MgCI2, 10 mM DTT, 500 gM each of dATP, dCTP, dGTP,
and dTTP, 50-250 J.tg/mL mRNA, either 25-125 ~tg/mL pd(T)12-18, 2-
10 gg/mL pd(N) 6, or 10-50 lag/mL 30 base primer-adapter, and 10,000-
50,000 U/mL SuperScript TM RT. - Place the tube on ice. Remove 2 pL from the reaction mixture, and add
them to 48 laL of 20 mM EDTA (pH 7.5) containing 5 ~tg of tRNA. This
will be used to calculate first-strand yield and to analyze the product by
gel electrophoresis (see Section 4.4.). - Use the remaining 18 I.tL of first-strand reaction mixture (or the entire
20-pL reaction mixture if 32p was not used) to carry out second-strand
synthesis.
4.3.2. One-Tube Double-Stranded cDNA Synthesis:
Second-Strand Reaction - This protocol is suitable for 1-5 lag mRNA originally in the 20-I.tL first-
strand reaction mixture. - On ice, add the following reagents in the order shown to the first-strand
reaction mixture tube: water to bring the final reaction mixture vol to
150 pL, 30 lxL 5X Second-Strand Buffer, 3 laL 10 mM dNTPs, 10 U E.
coli DNA ligase, 40 U E. coli DNA polymerase I, and 2 U E. coli RNase
H. If the second-strand product is to be labeled, add 10 ~tCi of [ot-
32p]dCTP after the unlabeled dNTPs. Vortex the tube gently, and incu-
bate at 16°C for 2 h. - The final composition is 25 mMTris-HC1 (pH 7.5),100 mM KCI, 5 mM
MgCI 2, 10 mM (NH4)2SO4, 0.15 mM ~-NAD, 250 ~4 each of dATP,
dCTP, dGTP, dTTP, 1.2 mM DTT, 67 U/mL DNA ligase, 267 U/mL
DNA polymerase I, and 13 U/mL RNase H. - Add 20 U of T4 DNA polymerase, and continue incubating at 16°C for
5 min. - Place the tube on ice. If [o~-32p]dCTP was added to the second-strand
reaction, remove 10 gL from the tube and add it to 40 pL of 20 mM
EDTA containing 5 lag of tRNA. This will be used to calculate yield
and to analyze the products by gel electrophoresis (Section 4.4.).