eDNA Synthesis with M-MLV RT 85
aqueous phase with 2.5 vol of ethanol. Dissolve the tRNA pellet in
0.4M NaCI-0.05M Tris-HCl, and repeat the precipitation. Dissolve the
pellet in sufficient autoclaved, distilled, deionized water to bring the
concentration to 1 mg/mL (17.7 A260 U/mg). Store the solution at -20°C.- Autoclaved, distilled water.
- Ethanol: 95% ethanol.
- Bovine serum albumin (BSA), 50 mg/mL.
4.2.2. Solutions- dNTPs (10 mM): Prepare 50-mM stocks of each dNTP by dissolving
13-15 mg of each sodium salt in 100 ~tL of H20. Adjust to pH 5-7 by
adding the appropriate amount of 1N NaOH (20-40 lxL). Use pH paper.
Add 4 ~L of 1M Tris-HC1 (pH 7.5) to buffer the solution, and bring the
volume of the solution to 500 ~tL with water. Check the concentration
of each solution at a 1/1000 dilution in water by determining the absor-
bance at 260 nm (E250 [1 crrgpH 7] for dCTP, dGTP, dATP, and dTTP
is 7.4, 12.8, 15.3, and 8.4 cm -l mM -1, respectively). Prepare a mixture
of all four dNTPs in water at a final concentration of 10 mM of each
dNTP. Store all solutions at -20°C. - 7.5 M Ammonium acetate.
- 0.1M Dithiothreitol (DTT) (store at-20°C).
- 0.5M EDTA: Adjust pH to 7.5 with NaOH.
- 5X First-Strand Buffer: 250 mM Tris-HC1 (pH 8.3 at room tempera-
ture), 375 mM KCI, and 15 mM MgCI 2 (store at -20°C). - 5X Second-Strand Buffer: 94 mM Tris-HCI (pH 6.9 at room tempera-
ture), 453 mM KCI, 23 mM MgC12, 750 pM ~-NAD, and 50 mM (NH4) 2
SO 4 (store at-20°C). - 10X DNA Synthesis Buffer: 200 mM Tris-HCl (pH 8.3 at room tempera-
ture), 500 mM KCI, 25 mM MgCI 2, and 1 mg/mL BSA (store at -20°C).
4.3. Model Protocols
4.3.1. One-Tube Double-Stranded cDNA Synthesis:
First-Strand Reaction - In a 1.5-mL tube, mix on ice 1-5 p.g poly(A) + mRNA and one of the
following:
- 0.5-2.5 lag pd(T)12_18
- 40-200 ng pd(N)6 or
- A 10-fold molar excess of a primer-adapter (0.2-1.0 pg pd[N]30 )
Add water to a vol of 7 pL; heat at 70°C for 10 min and chill on ice.
- In the tube on ice, mix 4 laL 5X First-Strand Buffer, 2 laL 0.1M DTT, 1 txL
10 mM dNTPs, 1 ktL [a-32p]dCTP (1 l.tCi/pL), and sufficient water to